技术领域technical field
本公开涉及，为了检测微生物以及在一些例子中鉴定微生物的目的，而对生物材料进行染色的方法。更具体地，本公开涉及用于在显微镜载玻片上实施自动化革兰氏染色的方法、组合物和试剂盒。The present disclosure relates to methods of staining biological materials for the purpose of detecting and, in some instances, identifying microorganisms. More specifically, the present disclosure relates to methods, compositions and kits for performing automated Gram stains on microscope slides.
背景background
革兰氏染色是一种公知的、普遍使用的微生物学技术，其用于微生物尤其是细菌的检测、分类和鉴定。革兰氏染色的最常见方法涉及四个步骤：染色、捕获、脱色和复染色。组织学实验室中常用的革兰氏染色已知是不稳定的，特别是由于脱色步骤中的波动所致。影响脱色步骤的变量包括溶剂强度、浓度、体积和暴露持续时间。在手动革兰氏染色过程中，脱色步骤常规使用醇或丙酮，且该步骤仅需数秒。然而，对于自动化染色过程，该脱色过程发生得太快。本公开针对在自动化染色平台上的革兰氏染剂脱色的问题。Gram staining is a well known and commonly used microbiological technique for the detection, classification and identification of microorganisms, especially bacteria. The most common method of Gram staining involves four steps: staining, capturing, destaining, and counterstaining. Gram stains commonly used in histology laboratories are known to be unstable, especially due to fluctuations in the destaining step. Variables that affect the decolorization step include solvent strength, concentration, volume, and duration of exposure. During manual Gram staining, alcohol or acetone is routinely used for the destaining step, and the step takes only seconds. However, this decolorization process occurs too quickly for an automated staining process. The present disclosure addresses the problem of Gram stain destaining on automated staining platforms.
概述overview
本文中提供了能自动进行革兰氏染色的方法、组合物和试剂盒。发明人已鉴定出延长革兰氏染色脱色步骤的组合物，其允许革兰氏染色的自动化，例如，使用自动化的和/或计算机执行的染色装备来实现自动化。Provided herein are methods, compositions and kits for automated Gram staining. The inventors have identified compositions that prolong the destaining step of the Gram stain that allow automation of the Gram stain, for example, using automated and/or computer-implemented staining equipment.
本文中提供了对样品(如在显微镜载玻片上的样品)进行革兰氏染色的方法。所述方法可用于检测和区分革兰氏阳性细菌和革兰氏阴性细菌。在一些例子中，所公开的革兰氏染色方法被用于检测并非细菌的微生物，如酵母(例如假丝酵母(Candida)和隐球酵母(Cryptococcus))。Provided herein are methods of Gram staining a sample, such as a sample on a microscope slide. The method can be used to detect and differentiate between Gram-positive and Gram-negative bacteria. In some instances, the disclosed Gram stain methods are used to detect microorganisms that are not bacteria, such as yeast (eg, Candida and Cryptococcus).
在具体的例子中，所述方法包括使样品与慢作用脱色制剂(如一种包含1，2-丙二醇或乙二醇的制剂)在足以从革兰氏阴性细菌而非革兰氏阳性细菌显著除去初级染剂-捕获剂复合物的条件下接触。所述样品是先前与初级染剂(如结晶紫)和捕获剂(如碘)接触过的样品。在具体的例子中，所述方法包括革兰氏染色过程的一个或多个另外的步骤，如使所述样品与初级染剂在足以将革兰氏阳性细菌和革兰氏阴性细菌的细胞壁和细胞膜染色的条件下接触；使所述样品与捕获剂在足以形成初级染剂-捕获剂复合物的条件下接触；使所述样品与复染剂在足以对已脱色的革兰氏阴性细菌进行染色的条件下接触，或其组合。In specific examples, the method comprises subjecting the sample to a slow-acting depigmenting formulation (such as one comprising 1,2-propanediol or ethylene glycol) at a temperature sufficient to significantly remove gram-negative but not gram-positive bacteria. contact under the conditions of the primary dye-capture agent complex. The sample is a sample that has been previously exposed to a primary stain (such as crystal violet) and a capture agent (such as iodine). In particular examples, the method includes one or more additional steps of the Gram staining process, such as exposing the sample to a primary stain sufficient to separate the cell walls of Gram-positive and Gram-negative bacteria and contacting the cell membrane under conditions that stain the cell membrane; contacting the sample with the capture agent under conditions sufficient to form a primary dye-capture agent complex; contacting the sample with a counterstain under conditions sufficient for destained Gram-negative bacteria Stained condition contact, or a combination thereof.
本文还提供能充当慢作用脱色制剂的组合物。在一个例子中，所述组合物包含至少80％的慢作用脱色剂(如1，2-丙二醇或乙二醇)和不超过20％的快作用脱色剂(如0.1-20％的快速作用脱色剂，例如乙醇或丙酮)。这类组合物可用作自动化革兰氏染色中的慢作用脱色制剂。Also provided herein are compositions that can act as slow acting depigmenting agents. In one example, the composition comprises at least 80% of a slow-acting depigmenting agent (such as 1,2-propylene glycol or ethylene glycol) and no more than 20% of a fast-acting depigmenting agent (such as 0.1-20% of a fast-acting depigmenting agent). solvents such as ethanol or acetone). Such compositions are useful as slow-acting depigmenting agents in automated Gram staining.
本文还提供用于自动化革兰氏染色的试剂盒。在一个例子中，所述试剂盒在容器中包含慢作用脱色制剂，如包含至少80％或至少90％的慢作用脱色剂(如1，2-丙二醇或乙二醇)和不超过20％的快作用脱色剂(如0.1-20％或1-10％的快速作用脱色剂，例如乙醇或丙酮)的制剂。在一些例子中，试剂盒可另外包含其他组分，如在容器中的一种或多种初级染剂；在容器中的捕获剂；在容器中的复染剂，显微镜载玻片，盖玻片，其他染剂，和对照载玻片。在具体的例子中，所述容器配置为与自动化革兰氏染色平台一起使用。Also provided herein are kits for automated Gram staining. In one example, the kit comprises a slow-acting depigmenting agent in a container, such as comprising at least 80% or at least 90% of a slow-acting depigmenting agent (such as 1,2-propylene glycol or ethylene glycol) and no more than 20% of A formulation of a fast-acting depigmenting agent such as 0.1-20% or 1-10% of a fast-acting depigmenting agent such as ethanol or acetone. In some examples, the kit may additionally contain other components such as one or more primary stains in a container; a capture reagent in a container; a counterstain in a container, microscope slides, coverslips slides, other stains, and control slides. In a specific example, the container is configured for use with an automated Gram staining platform.
本公开的前述和其他对象和特征将从下列详细描述并参照附图而变得更加明显。The foregoing and other objects and features of the present disclosure will become more apparent from the following detailed description taken with reference to the accompanying drawings.
附图简述Brief description of the drawings
图1是显示用于自动化革兰氏染色的例示性方法的流程图。Figure 1 is a flowchart showing an exemplary method for automated Gram staining.
详述detail
提供对术语和方法的以下解释以更好地描述本发明及指导本领域普通技术人员实施本发明。除非上下文清楚地另外指示，单数形式“一个”、“一种”和“该”指一种或超过一种。例如，术语“包含一个/一种标本”包括单数或复数标本，且视为等同于短语“包含至少一种标本”。除非上下文清楚地另外指示，术语“或”指所述备选要素中的单个要素或者指两个或更多个要素的组合。如本文中使用的，“包含”意指“包括”。如此，“包含A或B”意指“包含A、B或A和B”，而不排除别的要素。The following explanations of terms and methods are provided to better describe the invention and guide those of ordinary skill in the art to practice the invention. The singular forms "a", "an" and "the" mean one or more than one unless the context clearly dictates otherwise. For example, the term "comprising a specimen" includes singular or plural specimens and is considered equivalent to the phrase "comprising at least one specimen". Unless the context clearly dictates otherwise, the term "or" refers to a single of stated alternative elements or to a combination of two or more elements. As used herein, "comprises" means "includes". Thus, "comprising A or B" means "comprising A, B, or A and B", without excluding other elements.
除非另外解释，本文中使用的所有技术和科学术语均具有与本公开所述领域中的普通技术人员普遍理解的相同的含义。在有冲突的情况下，将以包括术语解释的本说明书为准。微生物学中的常见术语和方法的解释可见于Wistreich and Lechtman，Laboratory Exercises in Microbiology(微生物学的实验室手册)，第6版，1988和Boyd，General Microbiology(微生物学概述)，第2版，1988。Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In case of conflict, the present specification, including explanations of terms, will control. Explanations of common terms and methods in microbiology can be found in Wistreich and Lechtman, Laboratory Exercises in Microbiology, 6th edition, 1988 and Boyd, General Microbiology, 2nd edition, 1988 .
本文中提到的所有公开出版物、专利申请、专利和其他参考文献均通过提述完整并入以用于所有目的。All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety for all purposes.
尽管可将类似或等同于本文中描述的抗体和材料的那些用于实践或测试所公开的技术，但下文描述了适宜的方法和材料。所述材料、方法和例子仅为例示性的且不意图为限制的。Although antibodies and materials similar or equivalent to those described herein can be used in the practice or testing of the disclosed techniques, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and not intended to be limiting.
为了促进对本公开的各实施方案的评述，提供了对特定术语的以下解释：To facilitate the review of various embodiments of the present disclosure, the following explanations of certain terms are provided:
抗体(Ab)：包含至少一个轻链或重链免疫球蛋白可变区，且特异性结合抗原(如靶试剂)的表位的多肽。抗体包括单克隆抗体、多克隆抗体、或抗体片段以及本领域已知的其他抗体。在一些例子中，抗体特异于靶物，如具体细菌或细菌类型，如此可与本文中提供的方法相伴进行测定。Antibody (Ab): A polypeptide comprising at least one light or heavy chain immunoglobulin variable region, and which specifically binds an epitope of an antigen, such as a target agent. Antibodies include monoclonal antibodies, polyclonal antibodies, or antibody fragments as well as others known in the art. In some instances, antibodies are specific to a target, such as a particular bacterium or type of bacterium, and as such can be assayed in conjunction with the methods provided herein.
抗体由重链和轻链构成，它们各自具有可变区，称为可变重链(VH)区和可变轻链(VL)区。VH区和VL区一起负责结合抗体所识别的抗原。这包括完整的免疫球蛋白及本领域公知的它们的变体和组成部分，如Fab′片段、F(ab)′2片段、单链Fv蛋白(“scFv”)和被二硫键稳定的Fv蛋白(“dsFv”)。scFv蛋白是一种融合蛋白，其中免疫球蛋白的轻链可变区和免疫球蛋白的重链可变区由接头结合，而在dsFv中，各个链经突变引入了二硫键，从而稳定各个链的联合。该术语还包括重组形式如嵌合抗体(例如人源化的鼠抗体)和异偶联抗体(如双特异性抗体)。亦参见Pierce Catalog and Handbook，1994-1995(PierceChemical Co.，Rockford，IL)；Kuby，Immunology，第3版，W.H.Freeman&Co.，New York，1997。Antibodies are composed of heavy and light chains, each of which has variable regions, referred to as variable heavy (VH) and variable light (VL) regions. Together, the VH and VL regions are responsible for binding the antigen recognized by the antibody. This includes intact immunoglobulins as well as their variants and components known in the art, such as Fab' fragments, F(ab)'2 fragments, single chain Fv proteins ("scFv") and disulfide bond stabilized Fv protein ("dsFv"). The scFv protein is a fusion protein in which the light chain variable region of an immunoglobulin and the heavy chain variable region of an immunoglobulin are joined by a linker, whereas in dsFv the individual chains are mutated to introduce disulfide bonds, thereby stabilizing the individual chains. chain union. The term also includes recombinant forms such as chimeric antibodies (eg, humanized murine antibodies) and heteroconjugated antibodies (eg, bispecific antibodies). See also Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, Immunology, 3rd Edition, W.H. Freeman & Co., New York, 1997.
“单克隆抗体”是由B淋巴细胞的单个克隆或由转染了单个抗体的轻链和重链基因的细胞所产生。单克隆抗体由本领域技术人员已知的方法来产生，例如通过从骨髓瘤细胞与免疫脾细胞制备杂合的抗体形成细胞来产生。这些融合的细胞及其子代称作“杂交瘤”。单克隆抗体包括人源化的单克隆抗体。"Monoclonal antibodies" are produced by a single clone of B lymphocytes or by cells transfected with the light and heavy chain genes of a single antibody. Monoclonal antibodies are produced by methods known to those skilled in the art, for example, by making hybrid antibody-forming cells from myeloma cells and immune spleen cells. These fused cells and their progeny are called "hybridomas". Monoclonal antibodies include humanized monoclonal antibodies.
细菌：在一些例子中，原核生物导致疾病(病原菌)。根据其细胞壁的结构特征可将细菌分类。例如，“革兰氏阳性”细胞壁中多层厚厚的肽聚糖层染色呈紫色，而薄的“革兰氏阴性”细胞壁呈现粉色。通过将形态学和革兰氏染色组合，可将大部分细菌归属至四个组(革兰氏阳性球菌、革兰氏阳性杆菌、革兰氏阴性球菌和革兰氏阴性杆菌)。Bacteria: In some instances, prokaryotes cause disease (pathogens). Bacteria can be classified according to the structural features of their cell walls. For example, the thick layers of peptidoglycan in "Gram-positive" cell walls stain purple, while thin "Gram-negative" cell walls appear pink. By combining morphology and Gram staining, most bacteria can be assigned to four groups (Gram-positive cocci, Gram-positive bacilli, Gram-negative cocci, and Gram-negative bacilli).
可利用公开的革兰氏染色方法检测的细菌的例子，包括但不限于：鲍曼不动杆菌(Acinetobacter baumanii)、放线杆菌属物种(Actinobacillus sp.)、放射菌(Actinomycetes)、放线菌属物种(Actinomyces sp.)(如伊氏放线菌(Actinomyces israelii)和内氏放线菌(Actinomyces naeslundii))、气单胞菌属物种(Aeromonas sp.)(如嗜水气单胞菌(Aeromonas hydrophila)、维罗纳气单菌温和生物变种(Aeromonas veronii biovar sobria)(温和产气单胞菌(Aeromonas sobria)和豚鼠气单胞菌(Aeromonas caviae)、嗜吞噬细胞无形体(Anaplasma phagocytophilum)、木糖产碱杆菌(Alcaligenes xylosoxidans)、鲍曼不动杆菌(Acinetobacter baumanii)、伴放线放线杆菌(Actinobacillusactinomycetemcomitans)、杆菌属物种(Bacillus sp.)(如炭疽杆菌(Bacillusanthracis)、蜡样芽孢杆菌(Bacillus cereus)、枯草芽孢杆菌(Bacillus subtilis)、苏芸金杆菌(Bacillus thuringiensis)和嗜热脂肪芽孢杆菌(Bacillusstearothermophilus)、类杆菌属物种(Bacteroides sp.)(如脆弱类杆菌(Bacteroides fragilis)、巴尔通体菌属物种(Bartonella sp.)(如杆状巴尔通体(Bartonella bacilliformis)和汉赛巴尔通体(Bartonella henselae)、双歧杆菌属物种(Bifidobacterium sp.)、鲍特菌属物种(Bordetella sp.)(如百日咳鲍特菌(Bordetella pertussis)、副百日咳鲍特菌(Bordetella parapertussis)和支气管败血性鲍特菌(Bordetella bronchiseptica)、包柔氏螺旋体菌属物种(Borrelia sp.)(如回归热包柔体(Borrelia recurrentis)和伯氏包柔体(Borreliaburgdorferi))、布鲁氏菌属物种(Brucella sp.)(如流产布鲁氏菌(Brucellaabortus)、犬布鲁氏菌(Brucella canis)、羊布鲁氏菌(Brucella melintensis)和猪布鲁氏菌(Brucella suis))、伯克霍尔德氏菌属物种(Burkholderia sp.)(如类鼻疽伯克氏菌(Burkholderia pseudomallei)和洋葱伯克氏菌(Burkholderia cepacia)、弯曲菌属物种(Campylobacter sp.)(如空肠弯曲菌(Campylobacter jejuni)、大肠弯曲菌(Campylobacter coli)、红嘴鸥弯曲菌(Campylobacter lari)和胎儿弯曲菌(Campylobacter fetus))、二氧化碳噬纤维菌属物种(Capnocytophaga sp.)、人心杆菌(Cardiobacterium hominis)、沙眼衣原体(Chlamydia trachomatis)、肺炎嗜衣原体(Chlamydophilapneumoniae)、鹦鹉热衣原体(Chlamydophila psittaci)、柠檬酸杆菌属物种(Citrobacter sp.)、贝纳柯克斯体(Coxiella burnetii)、棒状杆菌属物种(Corynebacterium sp.)(比如，牛棒杆菌(Corynebacterium diphtheriae)、杰氏棒杆菌(Corynebacterium jeikeum)和棒状杆菌(Corynebacterium))、梭菌属物种(Clostridium sp.)(如产气荚膜梭菌(Clostridium perfringens)、艰难梭菌(Clostridium difficile)、肉毒梭菌(Clostridium botulinum)和破伤风梭菌(Clostridium tetani))、侵蚀艾肯菌(Eikenella corrodens)、肠杆菌属物种(Enterobacter sp.)(如产气肠杆菌(Enterobacter aerogenes)、成团肠杆菌(Enterobacter agglomerans)、阴沟肠杆菌(Enterobacter cloacae)和大肠杆菌(Escherichia coli)、包括条件性大肠杆菌(opportunistic Escherichia coli)，如肠产毒性大肠杆菌(enterotoxigenic E.Coli)，肠侵袭性大肠杆菌(enteroinvasive E.Coli)、肠致病性大肠杆菌(enteropathogenic E.Coli)、肠出血性大肠杆菌(enterohemorrhagic E.coli)、肠聚集性大肠杆菌(enteroaggregative E.Coli)和尿道致病性大肠杆菌(uropathogenic E.coli)肠球菌属物种(Enterococcus sp.)(如粪肠球菌(Enterococcus faecalis)和屎肠球菌(Enterococcus faecium)埃立克体属物种(Ehrlichia sp.)(如恰菲埃里希氏体(Ehrlichia chafeensia)和犬艾利希体(Ehrlichia canis))、猪丹毒杆菌(Erysipelothrix rhusiopathiae)、优杆菌属物种(Eubacterium sp.)、土拉热弗朗西氏菌(Francisella tularensis)、具核梭杆菌(Fusobacteriumnucleatum)、阴道嗜血杆菌(Gardnerella vaginalis)、麻疹孪生球菌(Gemellamorbillorum)、嗜血杆菌属物种(Haemophilus sp.)(如流感嗜血杆菌(Haemophilus influenzae)、杜克雷嗜血杆菌(Haemophilus ducreyi)、埃及嗜血杆菌(Haemophilus aegyptius)、副流感嗜血杆菌(Haemophilusparainfluenzae)、溶血嗜血杆菌(Haemophilus haemolyticus)和副溶血嗜血杆菌(Haemophilus parahaemolyticus)、螺杆菌属物种(Helicobacter sp.)(如幽门螺杆菌(Helicobacter pylori)、同性恋螺杆菌(Helicobacter cinaedi)和芬纳尔螺杆菌(Helicobacter fennelliae))、金氏金氏菌(Kingella kingii)、克雷伯氏菌属物种(Klebsiella sp.)(如克雷白氏肺炎菌(Klebsiella pneumoniae)、肉芽肿杆菌(Klebsiella granulomatis)和产酸克雷伯氏菌(Klebsiella oxytoca)，乳酸杆菌属物种(Lactobacillus sp.)、产单核细胞李斯特菌(Listeriamonocytogenes)、问号钩端螺旋体(Leptospira interrogans)、嗜肺性军团病杆菌(Legionella pneumophila)、问号钩端螺方定体(Leptospira interrogans)、消化链球菌属物种(Peptostreptococcus sp.)、粘膜炎莫拉菌(Moraxellacatarrhalis)、摩根氏菌属物种(Morganella sp.)、动弯杆菌属物种(Mobiluncussp.)、细球菌属物种(Micrococcus sp.)、分枝杆菌属种(Mycobacterium sp.)(如麻风分枝杆菌(Mycobacterium leprae)、结核分枝杆菌(Mycobacteriumtuberculosis)、胞内分枝杆菌(Mycobacterium intracellulare)、鸟分枝杆菌(Mycobacterium avium)、牛分枝杆菌(Mycobacterium bovis)和海分枝杆菌(Mycobacterium marinum))、支原菌属物种(Mycoplasm sp.)(如肺炎支原体(Mycoplasma pneumoniae)、人支原体(Mycoplasma hominis)和生殖器支原体(Mycoplasma genitalium))、诺卡氏菌属物种(Nocardia sp.)(如星形诺卡氏菌(Nocardia asteroides)、盖尔森基兴诺卡氏菌(Nocardiacyriacigeorgica)和巴西诺卡氏菌(Nocardia brasiliensis))、奈瑟氏菌属物种(Neisseria sp.)(如淋病奈瑟氏菌(Neisseria gonorrhoeae)和脑膜炎奈瑟氏菌(Neisseria meningitidis))、出血败血性巴斯德氏菌(Pasteurella multocidla)、类志贺氏邻单胞菌(Plesiomonas shigelloides)、普雷沃氏菌属物种(Prevotellasp.)、卟啉单胞菌属物种(Porphyromonas sp.)、产黑素普雷沃氏菌(Prevotellamelaninogenica)、变形菌属物种(Proteus sp.)(如普通变形菌(Proteusvulgaris)和奇异变形菌(Proteus mirabilis))、普罗威登斯菌属物种(Providencia sp.)(如产碱普罗威登斯菌(Providencia alcalifaciens)、雷氏普罗威登斯菌(Providencia rettgeri)和斯氏普罗威登斯菌(Providenciastuartii))、绿胺假单胞菌(Pseudomonas aeruginosa)、短小棒状杆菌(Propionibacterium acnes)、马红球菌(Rhodococcus equi)、立克次体属物种(Rickettsiasp.)(如立克氏立克次体(Rickettsia rickettsii)、螨立克次体(Rickettsia akari)和普氏立克次体(Rickettsia prowazekii)、恙虫病东方体(Orientia tsutsugamushi)(学名：恙虫病立克次体(Rickettsia tsutsugamushi)和地方性斑疫伤寒立克次氏体(Rickettsia typhi)、红球菌属物种(Rhodococcussp.)、粘质沙雷氏杆菌(Serratia marcescens)、嗜麦芽寡养单胞菌(Stenotrophomonas maltophilia)、沙门氏菌属物种(Salmonella sp.)(如肠道沙门氏菌(Salmonella enterica)、伤寒沙门氏菌(Salmonella typhi)、副伤寒沙门氏菌(Salmonella paratyphi)、肠炎沙门氏菌(Salmonella enteritidis)、猪霍乱沙门菌(Salmonella cholerasuis)和鼠伤寒沙门氏菌(Salmonellatyphimurium)、沙雷氏菌属物种(Serratia sp.)(如粘质沙雷氏菌(Serratiamarcesans)和液化沙雷氏菌(Serratia liquifaciens)、志贺氏菌属物种(Shigellasp.)(如志贺氏痢疾菌(Shigella dysenteriae)、弗氏志贺氏菌(Shigellaflexneri)、波伊德志贺氏菌(Shigella boydii)和宋内志贺菌(Shigella sonnei)、葡萄球菌属物种(Staphylococcus sp.)(如金黄色酿脓葡萄球菌(Staphylococcusaureus)、表皮葡萄球菌(Staphylococcus epidermidis)、溶血葡萄球菌(Staphylococcus hemolyticus)、腐生性葡萄球菌(Staphylococcussaprophyticus)、链球菌属物种(Streptococcus sp.)(如肺炎链球菌(Streptococcus pneumoniae)(例如氯霉素-抗性血清型4肺炎链球菌(chloramphenicol-resistant serotype 4 Streptococcus pneumoniae)、壮观霉素-抗性血清型6B炎链球菌(spectinomycin-resistant serotype 6B Streptococcuspneumoniae)、链霉素-抗性血清型9V肺炎链球菌(streptomycin-resistantserotype 9V Streptococcus pneumoniae)、红霉素-抗性血清型14肺炎链球菌(erythromycin-resistant serotype 14 Streptococcus pneumoniae)、奥普托欣-抗性血清型14肺炎链球菌(optochin-resistant serotype 14 Streptococcuspneumoniae)、利福平-抗性血清型18C肺炎链球菌(rifampicin-resistantserotype 18C Streptococcus pneumoniae)、四环素-抗性血清型19F肺炎链球菌(tetracycline-resistant serotype 19F Streptococcus pneumoniae)、青霉素-抗性血清型19F肺炎链球菌(penicillin-resistant serotype 19F Streptococcuspneumoniae)和甲氧苄氨嘧啶-抗性血清型23F肺炎链球菌(trimethoprim-resistant serotype 23F Streptococcus pneumoniae)、氯霉素-抗性血清型4肺炎链球菌(chloramphenicol-resistant serotype 4 Streptococcuspneumoniae，e 4 Streptococcus pneumoniae)、壮观霉素-抗性血清型6B肺炎链球菌(spectinomycin-resistant serotype 6B Streptococcus pneumoniae)、链霉素-抗性血清型9V肺炎链球菌(streptomycin-resistant serotype 9V Streptococcuspneumoniae)、奥普托欣-抗性血清型14肺炎链球菌(optochin-resistantserotype 14 Streptococcus pneumoniae)、利福平-抗性血清型18C肺炎链球菌(rifampicin-resistant serotype 18C Streptococcus pneumoniae)、青霉素-抗性血清型19F肺炎链球菌(penicillin-resistant serotype 19F Streptococcuspneumoniae)或甲氧苄氨嘧啶-抗性血清型23F肺炎链球菌(trimethoprim-resistant serotype 23F Streptococcus pneumoniae)、无乳链球菌(Streptococcus agalactiae)、变形链球菌(Streptococcus mutans)、化胺性链球菌(Streptococcus pyogenes)、A组链球菌(Group A streptococci)、化胺性链球菌(Streptococcus pyogenes)、B组链球菌属(Group B streptococci)、无乳链球菌(Streptococcus agalactiae)、C组链球菌属(Group C streptococci)、咽颊炎链球菌(Streptococcus anginosus)、类马链球菌(Streptococcusequismilis)、D组链球菌(Group D streptococci)、牛链球菌(Streptococcusbovis)、F组链球菌(Group F streptococci)和咽颊炎链球菌(Streptococcusanginosus)、G组链球菌(Group G streptococci)、鼠咬热螺旋体(Spirillumminus)、念珠状链杆菌(Streptobacillus moniliformi)、密螺旋体属物种(Treponema sp.)(如品他病密螺旋体(Treponema carateum)、细弱密螺旋体(Treponema petenue)、苍白密螺旋体(Treponema pallidum)和地方性密螺旋体(Treponema endemicum)、Tropheryma whippelii、解服支原体(Ureaplasma urealyticum)、韦荣球菌属物种(Veillonella sp.)、弧菌属物种(Vibrio sp.)(如霍乱弧菌(Vibrio cholerae)、副溶血性弧菌(Vibrioparahemolyticus)、嗜盐弧菌(Vibrio vulnificus)、副溶血性弧菌(Vibrioparahaemolyticus)、嗜盐弧菌(Vibrio vulnificus)、溶藻弧菌(Vibrioalginolyticus)、拟态弧菌(Vibrio mimicus)、霍氏弧菌(Vibrio hollisae)、河流弧菌(Vibrio fluvialis)、梅氏弧菌(Vibrio metchnikovii)、海鱼弧菌(Vibriodamsela)和弗氏弧菌(Vibrio furnisii)、耶尔森氏菌属物种(Yersinia sp.)(如小肠结肠炎耶尔森氏菌(Yersinia enterocolitica)、鼠疫耶尔森氏菌(Yersiniapestis)和假结核耶尔森氏菌(Yersinia pseudotuberculosis)和嗜麦芽黄单胞菌(Xanthomonas maltophilia)等。Examples of bacteria that can be detected using published Gram stain methods include, but are not limited to: Acinetobacter baumanii, Actinobacillus sp., Actinomycetes, Actinomycetes Actinomyces sp. (such as Actinomyces israelii and Actinomyces naeslundii), Aeromonas sp. (such as Aeromonas hydrophila ( Aeromonas hydrophila), Aeromonas veronii biovar sobria (Aeromonas sobria) and Aeromonas caviae, Anaplasma phagocytophilum , Alcaligenes xylosoxidans, Acinetobacter baumanii, Actinobacillus actinomycetemcomitans, Bacillus sp. (such as Bacillus anthracis, Bacillus cereus Bacillus cereus, Bacillus subtilis, Bacillus thuringiensis and Bacillus stearothermophilus, Bacteroides sp. (such as Bacteroides fragilis , Bartonella sp. (such as Bartonella bacilliformis and Bartonella henselae), Bifidobacterium sp., Bordetella sp. .) (eg, Bordetella pertussis, Bordetella parapertussis, and Bordetella bronchiseptica, Borrelia sp. (eg, relapsing fever Borrelia recurrentis and Borrelia recurrentis Borreliaburgdorferi), Brucella sp. (such as Brucella abortus, Brucella canis, Brucella melintensis and pig cloth Brucella suis), Burkholderia sp. (such as Burkholderia pseudomallei and Burkholderia cepacia), Campylobacter sp. ( Campylobacter sp.) (eg, Campylobacter jejuni, Campylobacter coli, Campylobacter lari, and Campylobacter fetus), Capnocytophaga sp. .), Cardiobacterium hominis, Chlamydia trachomatis, Chlamydophila pneumoniae (Chlamydophila neumoniae), Chlamydophila psittaci (Chlamydophila psittaci), Citrobacter sp. Coxiella burnetii), Corynebacterium sp. (e.g., Corynebacterium diphtheriae, Corynebacterium jeikeum, and Corynebacterium), Clostridium sp. ( Such as Clostridium perfringens (Clostridium perfringens, Clostridium difficile, Clostridium botulinum and Clostridium tetani), Eikenella corrodens, Enterobacter Enterobacter sp. (such as Enterobacter aerogenes, Enterobacter agglomerans, Enterobacter cloacae, and Escherichia coli li), including opportunistic Escherichia coli, such as enterotoxigenic E.Coli, enteroinvasive E.Coli, enteropathogenic E.Coli ), enterohemorrhagic E.coli, enteroaggregative E.Coli, and uropathogenic E.coli Enterococcus sp. (such as fecal Enterococcus faecalis and Enterococcus faecium Ehrlichia sp. (eg Ehrlichia chafeensia and Ehrlichia canis), Erysipelothrix rhusiopathiae, Eubacterium sp., Francisella tularensis, Fusobacterium nucleatum, Gardnerella vaginalis, measles twins Gemella morbillorum, Haemophilus sp. (such as Haemophilus influenzae, Haemophilus ducreyi, Haemophilus aegyptius, Haemophilus parainfluenzae ( Haemophilus parainfluenzae), Haemophilus haemolyticus and Haemophilus parahaemolyticus, Helicobacter sp. (such as Helicobacter pylori, Helicobacter cinaedi and Fen Helicobacter fennelliae), Kingella kingii, Klebsiella sp. (such as Klebsiella pneumoniae ), Klebsiella granulomatis and Klebsiella oxytoca, Lactobacillus sp., Listeriamonocytogenes, Leptospira interrogans ), Legionella pneumophila, Leptospira interrogans, Peptostreptococcus sp., Moraxella catarrhalis, Morganella spp. (Morganella sp.), Mobiluncus sp., Micrococcus sp., Mycobacterium sp. (such as Mycobacterium leprae, Mycobacterium tuberculosis Mycobacterium tuberculosis, Mycobacterium intracellulare, Mycobacterium avium, Mycobacterium bovis and Mycobacterium marinum), Mycoplasma species sp.) (eg, Mycoplasma pneumoniae, Mycoplasma hominis, and Mycoplasma genitalium), Nocardia sp. (eg, Nocardia asteroides , Nocardiacyriacigeorgica, and Nocardia brasiliensis), Neisseria sp. (eg, Neisseria gonorrhoeae, and meningitis Neisseria meningitidis), Pasteurella multocidla, Plesiomonas shigeloides, Prevotella sp., porphyrins Porphyromonas sp., melanogenic Prevotella melaninogenica, Proteus sp. (such as Proteus vulgaris and Proteus mirabilis), Providencia sp. (such as Providencia alcalifaciens, Providencia rettgeri and Providenciastuartii), Pseudomonas aeruginosa, Corynebacterium pumilus (Propionibacterium acnes), Rhodococcus equi, Rickettsia sp. (such as Rickettsia rickettsii, Rickettsia akari, and Rickettsia sp. Rickettsia prowazekii, Orientia tsutsugamushi (scientific name: Rickettsia tsutsugamushi) and endemic Rickettsia typhi, Rhodococcus species ( Rhodococcus sp.), Serratia marcescens, Stenotrophomonas maltophilia, Salmonella sp. (such as Salmonella enterica, Salmonella typhi ), Salmonella paratyphi, Salmonella enteritidis, Salmonella cholerasuis and Salmonella typhimurium, Serratia sp. (such as Serratia marcescens Serratia marcesans and Serratia liquifaciens, Shigella sp. (eg Shigella dysenteriae, Shigella flexneri, Boey Shigella boydii and Shigella sonnei ( Shigella sonnei), Staphylococcus sp. (such as Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus hemolyticus, Staphylococcus saprophyticus, streptococcus Streptococcus sp. (eg, Streptococcus pneumoniae (eg, chloramphenicol-resistant serotype 4 Streptococcus pneumoniae), spectinomycin-resistant serotype 6B Streptococcus pneumoniae (spectinomycin-resistant serotype 6B Streptococcus pneumoniae), streptomycin-resistant serotype 9V Streptococcus pneumoniae, erythromycin-resistant serotype 14 Streptococcus pneumoniae 14 Streptococcus pneumoniae), optochin-resistant serotype 14 Streptococcus pneumoniae, rifampicin-resistant serotype 18C Streptococcus pneumoniae, tetracycline- Tetracycline-resistant serotype 19F Streptococcus pneumoniae, penicillin-resistant serotype 19F Streptococcus pneumoniae, and trimethoprim-resistant serotype 23F Streptococcus pneumoniae Trimethoprim-resistant serotype 23F Streptococcus pneumoniae, chloramphenicol-resistant serotype 4 Streptococcus pneumoniae 4 Streptococcus pneumoniae, e 4 Streptococcus pneumoniae), spectinomycin-resistant serotype 6B Streptococcus pneumoniae, streptomycin-resistant serotype 9V Streptococcus pneumoniae ), optochin-resistant serotype 14 Streptococcus pneumoniae, rifampicin-resistant serotype 18C Streptococcus pneumoniae, penicillin-resistant Penicillin-resistant serotype 19F Streptococcus pneumoniae or trimethoprim-resistant serotype 23F Streptococcus pneumoniae, Streptococcus agalactiae, strain Streptococcus mutans, Streptococcus pyogenes, Group A streptococci, Streptococcus pyogenes, Group B streptococci, Streptococcus agalactiae Streptococcus agalactiae, Group C streptococci, Streptococcus anginosus, Streptococcus equisilis, Group D streptococci, Streptococcus bovis ), Group F streptococci (Group F streptococci) and pharyngeal bucc streptococci (Streptococcus anginosus), G group streptococci (Group G streptococci), rat-bite Thermospira (Spirillumminus), Streptococcus moniliforme (St reptobacillus moniliformi), Treponema sp. (such as Treponema carateum, Treponema petenue, Treponema pallidum, and Treponema endemicum, Tropheryma whippelii, Ureaplasma urealyticum, Veillonella sp., Vibrio sp. (such as Vibrio cholerae, Vibrioparahemolyticus, Vibrio vulnificus, Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio alginolyticus, Vibrio mimicus, Vibrio hollisae ), Vibrio fluvialis, Vibrio metchnikovii, Vibriodamsela and Vibrio furnisii, Yersinia sp. (e.g. small intestinal Yersinia enterocolitica, Yersinia pestis, Yersinia pseudotuberculosis and Xanthomonas maltophilia etc.
接触：直接的物理结合；包括固体形式和液体形式两种。例如，接触可在体外发生于试剂(如革兰氏染料中的一个成分)和样品之间，所述样品位于溶液中或一个表面上(例如在显微镜载玻片上)。Contact: Immediate physical association; includes both solid and liquid forms. For example, contact can occur in vitro between a reagent (eg, a component of a Gram dye) and a sample, either in solution or on a surface (eg, on a microscope slide).
脱色剂(decolorizing agent)：可从样品(利如用初级染剂和捕获剂染色的样品)中基本上去除初级染剂(例如结晶紫)和捕获剂(例如碘)复合物的试剂，比如从革兰氏阴性细菌而非革兰氏阳性细菌的细胞壁中基本上去除所述复合物的试剂。在一个例子中，这样的复合物称作结晶紫-碘(CV-I)复合物。在具体的例子中，脱色剂与含有初级染剂-捕获剂复合物(例如CV-I复合物)的样品一起孵育一段时间，所述时间足以从革兰氏阴性细胞而非革兰氏阳性细胞中基本上去除(比如去除至少90％、至少95％或至少99％的)所述复合物(且从而去除初级染剂)。decolorizing agent: An agent that substantially removes a complex of a primary stain (e.g., crystal violet) and a capture agent (e.g., iodine) from a sample (e.g., a sample stained with a primary stain and a capture agent), such as from An agent that substantially removes the complex from the cell wall of Gram-negative bacteria but not Gram-positive bacteria. In one example, such a complex is called a crystal violet-iodine (CV-I) complex. In a specific example, the depigmenting agent is incubated with a sample containing a primary stain-capture complex (e.g., a CV-I complex) for a period of time sufficient to destain from Gram-negative cells but not Gram-positive cells. The complex (and thereby the primary stain) is substantially removed (eg, at least 90%, at least 95%, or at least 99%) is removed.
脱色制剂(decolorizing formulation)：组合物，包括一种或多种脱色剂。慢作用脱色剂是需要更长的时间才能从革兰氏阴性细胞中去除初级染剂-捕捉剂(例如，CV-I)复合物(且从而去除初级染剂，例如，结晶紫)的一种试剂，所述时间例如是，允许自动化设备完成脱色步骤、从而维持该自动化设备在完成所需脱色后将要发生另外的脱色作用(比如使革兰氏阳性细菌脱色)之前控制脱色步骤的完结的能力所需的时间。在一些例子中，缓慢脱色的时间是至少30秒、至少60秒、至少120秒、或至少220秒，例如1至4分钟，以从革兰氏阴性细菌中去除初级染剂-捕获剂(例如CV-I)复合物。慢作用脱色剂的例子包括1，2-丙二醇和乙二醇。快作用脱色剂是仅需要短时间就能从革兰氏阴性细胞中去除初级染剂-捕获剂(例如CV-I)复合物(且从而去除初级染剂，例如结晶紫)的一种试剂，所述时间比如少于20秒、少于15秒、少于10秒、或少于5秒，例如1至2秒、1至5秒、1至10秒或1至20秒。例子包括乙醇、丙酮、二乙醚、甲醚，乙醇与丙酮的共混物，以及乙醇与其他醇类如甲醇或2-丙醇的共混物。decolorizing formulation: A composition comprising one or more decolorizing agents. A slow-acting depigment is one that takes longer to remove the primary stain-capture (e.g., CV-I) complex (and thus the primary stain, e.g., crystal violet) from Gram-negative cells Reagents, such as times that allow automated equipment to complete the decolorization step, thereby maintaining the ability of the automated equipment to control the completion of the decolorization step prior to additional decolorization (such as decolorization of Gram-positive bacteria) following completion of the desired decolorization the time required. In some examples, the time for slow destaining is at least 30 seconds, at least 60 seconds, at least 120 seconds, or at least 220 seconds, such as 1 to 4 minutes, to remove the primary stain-capture agent (such as CV-I) complex. Examples of slow acting depigmenting agents include 1,2-propanediol and ethylene glycol. A fast-acting depigment is an agent that requires only a short time to remove the primary stain-capture (e.g. CV-I) complex (and thus the primary stain, e.g. crystal violet) from Gram-negative cells, The time is, for example, less than 20 seconds, less than 15 seconds, less than 10 seconds, or less than 5 seconds, such as 1 to 2 seconds, 1 to 5 seconds, 1 to 10 seconds or 1 to 20 seconds. Examples include ethanol, acetone, diethyl ether, methyl ether, blends of ethanol and acetone, and blends of ethanol with other alcohols such as methanol or 2-propanol.
检测：为确定一种因素(如信号或具体生物，如革兰氏阳性或革兰氏阴性细菌)是否存在，例如具体细菌。在一些例子中，这可进一步包括定量。例如，本文公开的方法和试剂在具体例子中的应用允许报告出较强和较弱的信号。Detection: To determine the presence or absence of an agent such as a signal or a specific organism such as Gram-positive or Gram-negative bacteria, such as specific bacteria. In some instances, this may further include quantification. For example, application of the methods and reagents disclosed herein in specific instances allows for the reporting of both stronger and weaker signals.
革兰氏阳性细菌：在革兰氏染色过程中染色深蓝色或紫色的细菌，且具有厚的肽聚糖层。革兰氏阳性细菌的例子包括：Gram-Positive Bacteria: Bacteria that stain dark blue or purple during Gram staining and have a thick layer of peptidoglycan. Examples of Gram-positive bacteria include:
革兰氏阴性细菌：在革兰氏染色过程中染色较浅或不能维持深蓝色或紫色，而是被复染剂如番红着色并呈现粉色或红色的细菌。革兰氏阴性细菌具有薄的肽聚糖层。示例性的革兰氏阴性细菌包括：Gram-negative bacteria: Bacteria that stain lightly or fail to maintain a dark blue or purple color during a Gram stain, but instead are stained pink or red by a counterstain such as safranin. Gram-negative bacteria have a thin peptidoglycan layer. Exemplary Gram-negative bacteria include:
初级染剂：在革兰氏染色中，其是应用于样品的第一种染料。初级染剂的例子是结晶紫染料。Primary Stain: In Gram staining, it is the first dye applied to a sample. An example of a primary dye is crystal violet dye.
定量：确定或测量靶标的量(如相对量)，如存在于样品中的靶细菌的量。Quantitation: Determining or measuring the amount (eg, relative amount) of a target, such as the amount of target bacteria present in a sample.
参考值：代表具体状况的数值或数值范围。实验值可以与参考值相比较，例如以进行诊断或预后。例如，参考值可以是期望来定义阳性或阴性结果(如革兰氏阳性或革兰氏阴性)的颜色的相对的或绝对的、最大的或最小的量(或范围)。Reference value: A value or range of values representing a specific situation. Experimental values can be compared to reference values, for example for diagnosis or prognosis. For example, a reference value may be a relative or absolute, maximum or minimum amount (or range) desired to define a color of a positive or negative result (eg, Gram positive or Gram negative).
载玻片：通常用于装载或固定样品以用于显微镜检的基片，其通常是透明至浅色的，但不必须如此。样品可在装载到载玻片上之前和/或之后进行处理。在一些例子中，载玻片可以是差不多透明的或不透明的，且由玻璃、二氧化硅、石英或适应于革兰氏染色的任何其他材料制成。可将载玻片配置成容纳来自一个或多个受试者的一个或多个样品。如本文所用的载玻片还包括非常规的基片，比如条带、圆盘、平板，或适合于呈现用于革兰氏染色的样品的任何其他扁平的、弯曲的、矩形的或球形的表面或形状。Slide: A substrate, usually, but not necessarily clear to lightly colored, usually used to mount or hold samples for microscopy. Samples can be processed before and/or after loading onto slides. In some examples, slides can be nearly transparent or opaque, and made of glass, silica, quartz, or any other material suitable for Gram staining. A slide can be configured to hold one or more samples from one or more subjects. Slides as used herein also include unconventional substrates such as strips, disks, plates, or any other flat, curved, rectangular or spherical shape suitable for presenting samples for Gram staining surface or shape.
受试者：活的或死亡的多细胞生物，包括植物以及人类和非人动物，比如牲畜受试者(例如，鸟、猫、狗、奶牛、猪、马和啮齿动物)。在一个例子中，受试者已知有或怀疑有细菌感染。Subject: Living or dead multicellular organisms, including plants and human and non-human animals, such as livestock subjects (eg, birds, cats, dogs, cows, pigs, horses, and rodents). In one example, the subject is known or suspected to have a bacterial infection.
捕获剂：与染料形成复合物且然后降低或减缓可能从样品或底物中洗掉染料的程度的试剂。在革兰氏染色方案中，碘是示例的捕获剂。捕获剂有时被称作媒染剂，但更精确地是捕获剂。媒染剂与底物更紧密地结合且不容易洗掉，并且，在革兰氏染色中，一些会被清洗去除的能力其实是为避免假阳性的目的而需要的。媒染剂与染料形成复合物，该复合物随后再与底物(比如织物)结合，然后有效避免了洗去染料。捕获剂也与染料形成复合物，但该复合物仅阻止洗的过程中染料的去除。捕获作用的机制通常是未知的，但可能归因于有益的扩散阻碍或空间阻碍。捕获剂一般不会通过与底物的强力键接起作用，与捕获剂一起形成的复合物中的染料仍可能在适宜的条件下被去除，比如，如在革兰氏染色中，通过足够有效的脱色剂的足够长的应用来去除。Capture Reagent: A reagent that forms a complex with a dye and then reduces or slows down the extent to which the dye may be washed out of the sample or substrate. In Gram staining protocols, iodine is an exemplary capture agent. Capture agents are sometimes called mordants, but more precisely capture agents. The mordant binds more tightly to the substrate and does not wash off easily, and, in Gram staining, some of the ability to be washed away is actually required for the purpose of avoiding false positives. The mordant forms a complex with the dye which then binds to the substrate (eg fabric) and effectively prevents the dye from being washed out. The capture agent also forms a complex with the dye, but this complex only prevents the removal of the dye during washing. The mechanism of trapping is generally unknown but may be attributed to beneficial diffusion or steric hindrance. Capture agents generally do not act by strong binding to the substrate, and dyes in complexes formed with capture agents may still be removed under suitable conditions, e.g., as in Gram staining, by sufficiently efficient A sufficiently long application of the depigmentation agent to remove.
在足以......的条件下：该短语用于描述允许所需活性的任何环境。这包括使试剂与样品在一定条件下相接触，所述条件允许所述试剂：在物理上或化学上与所述样品相互作用或反应、在所述样品中扩散、或置换出所述样品中的其他组分。在一个具体例子中，这包括将样品(比如一种已知含有或怀疑含有细菌的样品)与革兰氏染色的一种或多种试剂或组分相接触，以允许在样品中精确检测革兰氏阴性或革兰氏阳性。Under conditions sufficient to: This phrase is used to describe any circumstances that permit the desired activity. This includes contacting a reagent with a sample under conditions that allow the reagent to: physically or chemically interact or react with, diffuse within, or displace from, the sample of other components. In a specific example, this involves contacting a sample (such as a sample known or suspected to contain bacteria) with one or more reagents or components of a Gram stain to allow accurate detection of Gram stains in the sample. gram-negative or gram-positive.
总述Overview
革兰氏染色是熟知的，常用于微生物技术以用于微生物尤其细菌的检测、分类和鉴定，比如基于其细胞膜结构将细菌分为两大类：革兰氏阳性和革兰氏阴性。革兰氏阳性和革兰氏阴性细菌之间的首要区别在于，在革兰氏阳性细菌中，初级染剂吸收于整个细胞结构中，而在革兰氏阴性细菌中，染色仅发生在表面。因此，当随后利用脱色剂处理样品时，革兰氏阴性细菌倾向于失去其颜色，而革兰氏阳性细菌通常保持染色的蓝色或紫色。Gram stains are well known and commonly used in microbiological techniques for the detection, classification and identification of microorganisms, especially bacteria, such as dividing bacteria into two groups based on their cell membrane structure: Gram-positive and Gram-negative. The first difference between gram-positive and gram-negative bacteria is that in gram-positive bacteria, the primary stain is absorbed throughout the cellular structure whereas in gram-negative bacteria, the staining occurs only at the surface. Thus, when the sample is subsequently treated with a depigmenting agent, Gram-negative bacteria tend to lose their color, while Gram-positive bacteria typically remain stained blue or purple.
革兰氏染色比较容易有不一致的和波动的结果。尤其是，脱色步骤可能在使细菌适当脱色方面存在问题。若脱色不足，革兰氏阴性细菌保留初级染剂(例如，结晶紫)，而若过度脱色，革兰氏阳性细菌和革兰氏阴性细菌均会褪色。脱色中的不一致性可归因于初级染剂-捕捉剂复合物对脱色溶剂的亲和力、脱色溶剂的用量、脱色溶剂的相对溶解强度，以及脱色溶剂与样品接触的时间。Gram stains are relatively prone to inconsistent and fluctuating results. In particular, the decolorization step can be problematic in properly decolorizing the bacteria. With insufficient depigmentation, Gram-negative bacteria retain the primary stain (eg, crystal violet), whereas with excessive depigmentation, both Gram-positive and Gram-negative bacteria fade. Inconsistencies in destaining can be attributed to the affinity of the primary stain-capturer complex for the destaining solvent, the amount of destaining solvent used, the relative solubility strength of the destaining solvent, and the time the destaining solvent is in contact with the sample.
在常规人工革兰氏染色方法中，脱色步骤仅需要少量体积(比如2-3滴)，且仅花费几秒来使革兰氏阴性细菌脱色。革兰氏染色按常规制备并人工分析。然而，这对于自动化的程序而言太快了，因为仪器的限制可能在该步骤需要几分钟。结果，当在自动化的系统中使用常规的脱色剂时，发生不可接受的程度的脱色。因此，本文提供新的慢作用脱色剂以及能延长脱色步骤并允许自动化革兰氏染色的制剂。In conventional artificial Gram staining methods, the destaining step requires only a small volume (say 2-3 drops) and takes only a few seconds to destain the Gram-negative bacteria. Gram stains were routinely prepared and analyzed manually. However, this is too fast for an automated procedure as this step may take several minutes due to instrument limitations. As a result, unacceptable levels of decolorization occur when conventional depigmentation agents are used in automated systems. Accordingly, provided herein are new slow-acting depigmenting agents as well as formulations that prolong the depigmentation step and allow automated Gram staining.
革兰氏染色的方法Gram staining method
传统上，革兰氏染色包括4个步骤，各步骤对样品施用以下试剂之一：初级染剂，常见是结晶紫染料；捕获剂(有时称为媒染剂)，常见为碘；脱色剂，常见为乙醇或乙醇/丙酮；和复染剂，通常含有番红精O、品红或其他红色染料。本公开提供在包括非常快速脱色步骤(例如1-5秒)的革兰氏染色的现有手动方法上进行改进的方法。所公开的用于革兰氏染色的方法可用于自动化过程，因为已鉴定出延长脱色步骤的慢作用脱色剂(例如脱色花费至少30秒或至少60秒)。Traditionally, Gram staining consists of four steps, each of which applies one of the following reagents to the sample: a primary stain, usually crystal violet dye; a capture agent (sometimes called a mordant), usually iodine; and a depigmenting agent, usually is ethanol or ethanol/acetone; and a counterstain, usually containing safranin O, magenta, or other red dyes. The present disclosure provides methods that improve upon existing manual methods of Gram staining that include a very rapid destaining step (eg, 1-5 seconds). The disclosed methods for Gram staining can be used in automated processes, as slow-acting depigmenting agents have been identified that prolong the depigmentation step (eg depigmentation takes at least 30 seconds or at least 60 seconds).
本公开内容提供对样品进行革兰氏染色的方法。所述方法可用于检测和区分样品中存在的革兰氏阳性和革兰氏阴性细菌。在具体的例子中，所述方法包括使一种样品(或多种样品)与慢作用脱色制剂在足以从革兰氏阴性细菌而非革兰氏阳性细菌除去初级染剂-捕获剂复合物(例如CV-I复合物)的条件下接触，其中所述样品先前与初级染剂和捕获剂接触过。The present disclosure provides methods of Gram staining a sample. The method can be used to detect and differentiate between Gram-positive and Gram-negative bacteria present in a sample. In a specific example, the method comprises subjecting a sample (or samples) to a slow-acting depigmenting formulation at a temperature sufficient to remove the primary stain-capture complex from Gram-negative bacteria but not Gram-positive bacteria ( For example, under conditions of CV-I complex), wherein the sample has been previously contacted with primary stain and capture agent.
将领会在一些例子中，所述方法进一步包括革兰氏染色的一个或多个其他步骤。例如，该方法还可以包括以下一或多项：使样品与初级染剂在足以将革兰氏阳性和革兰氏阴性细菌的细胞壁(其包含肽聚糖)(且在一些例子中细胞膜)染色的条件下接触，使样品与捕获剂在足以形成初级染剂-捕获剂复合物的条件下接触，这可以阻止从革兰氏阳性细菌而非革兰氏阴性细菌除去初级染剂，以及使样品与复染剂在足以将脱色的革兰氏阴性细菌的细胞壁染色(其包含肽聚糖)(且在一些例子中细胞膜)的条件下接触。It will be appreciated that in some instances, the method further includes one or more additional steps of Gram staining. For example, the method may also include one or more of the following: subjecting the sample to the primary stain at a temperature sufficient to stain the cell walls (which comprise peptidoglycan) (and in some instances, cell membranes) of Gram-positive and Gram-negative bacteria. contacting the sample with the capture agent under conditions sufficient to form a primary stain-capture agent complex, which prevents the removal of the primary stain from Gram-positive but not Gram-negative bacteria, and allows the sample to Contact with a counterstain under conditions sufficient to stain the cell wall (which contains peptidoglycan) (and in some instances the cell membrane) of the depigmented Gram-negative bacteria.
使样品与初级染剂接触可包括使样品与包含结晶紫(CV)或甲紫(Gentianviolet)的制剂接触或将样品与其温育。在一个例子中，初级染剂制剂包含浓度为至少0.5g/L、至少0.75g/L、至少1g/L、至少2g/L、至少3g/L、至少4g/L，或至少5g/L，如1-4g/L，例如3g/L的CV或甲紫。在一些例子中，CV或甲紫存在于含有异丙醇、乙醇/甲醇和水(如50ml异丙醇、50ml乙醇/甲醇和900ml水)的溶液中。在一些例子中，所述CV或甲紫溶液含有草酸铵以增强稳定性。Contacting the sample with the primary stain can comprise contacting or incubating the sample with a formulation comprising crystal violet (CV) or Gentian violet. In one example, the primary dye formulation comprises a concentration of at least 0.5 g/L, at least 0.75 g/L, at least 1 g/L, at least 2 g/L, at least 3 g/L, at least 4 g/L, or at least 5 g/L, Such as 1-4g/L, such as 3g/L of CV or methyl violet. In some instances, CV or violet is present in a solution containing isopropanol, ethanol/methanol, and water (eg, 50 ml isopropanol, 50 ml ethanol/methanol, and 900 ml water). In some instances, the CV or violet solution contains ammonium oxalate to enhance stability.
使样品与捕获剂接触可包括使样品与包含碘的制剂如革兰氏碘或聚乙烯吡咯烷酮-碘(PVP-碘)溶液接触或将样品与其温育。革兰氏碘是一种混合物，其可以包含至少0.1％碘(例如至少0.2％、至少0.5％，或至少1％，如约0.1％-1％碘)和至少0.1％碘化钾(例如至少0.2％、至少0.5％、至少1％、至少1.5％，或至少2％，如约0.1％-2％碘化钾)，例如在水或醇混合物中。PVP-碘包含在水中至少1％聚乙烯吡咯烷酮(例如至少2％、至少5％、至少10％、至少20％、至少30％，或至少35％，如约1-35％聚乙烯吡咯烷酮)和至少1％碘(例如至少2％、至少5％、至少10％、至少20％、至少30％，或至少35％，如约1％-35％碘)(例如参见美国专利第2,739,922号和第3,898,326号)。Contacting the sample with a capture agent can include contacting or incubating the sample with a formulation comprising iodine, such as Gram's iodine or a polyvinylpyrrolidone-iodine (PVP-iodine) solution. Gram iodine is a mixture which may comprise at least 0.1% iodine (e.g. at least 0.2%, at least 0.5%, or at least 1%, such as about 0.1%-1% iodine) and at least 0.1% potassium iodide (e.g. at least 0.2%, At least 0.5%, at least 1%, at least 1.5%, or at least 2%, such as about 0.1% to 2% potassium iodide), for example in water or an alcoholic mixture. PVP-iodine comprises at least 1% polyvinylpyrrolidone (e.g., at least 2%, at least 5%, at least 10%, at least 20%, at least 30%, or at least 35%, such as about 1-35% polyvinylpyrrolidone) and at least 1% iodine (e.g., at least 2%, at least 5%, at least 10%, at least 20%, at least 30%, or at least 35%, such as about 1%-35% iodine) (see, e.g., U.S. Pat. Nos. 2,739,922 and 3,898,326 ).
使样品与复染剂接触可包括使样品与包含品红(如碱性品红或石炭酸品红)、中性红、和/或番红精O的制剂接触或将样品与其温育。在一个例子中，所述复染剂制剂包含浓度为至少0.01％、至少0.02％、至少0.03％、至少0.05％、至少0.1％、至少0.2％、至少0.3％、至少0.4％，或至少0.5％，如0.02％至0.5％的品红。在一个例子中，所述复染剂制剂包含浓度为至少0.01％、至少0.02％、至少0.03％、至少0.05％、至少0.1％、至少0.2％、至少0.3％、至少0.4％，或至少0.5％，如0.02％至1％的中性红。在一个例子中，所述复染剂制剂包含浓度为至少0.1％，如至少0.2％、至少0.3％、至少0.4％、至少0.5％、至少0.6％、至少0.7％，或至少0.8％，如0.1％至1％或0.1％至0.8％的番红精O。复染剂可以配制在水中，但在一些例子中可包含1％-20％乙醇或甲醇。Contacting the sample with a counterstain can include contacting or incubating the sample with a formulation comprising fuchsin (eg, basic fuchsin or carbofuran), neutral red, and/or safranin O. In one example, the counterstain formulation comprises a concentration of at least 0.01%, at least 0.02%, at least 0.03%, at least 0.05%, at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, or at least 0.5% , such as 0.02% to 0.5% magenta. In one example, the counterstain formulation comprises a concentration of at least 0.01%, at least 0.02%, at least 0.03%, at least 0.05%, at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, or at least 0.5% , such as 0.02% to 1% neutral red. In one example, the counterstain formulation comprises a concentration of at least 0.1%, such as at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, or at least 0.8%, such as 0.1 % to 1% or 0.1% to 0.8% Safranin O. Counterstains can be formulated in water, but in some instances may contain 1%-20% ethanol or methanol.
在一些例子中，将样品与慢作用脱色制剂和复染剂同时温育。在一些例子中，慢作用脱色制剂在慢作用脱色制剂/复染色制剂组合中的浓度范围可从约75％至99％，如至少75％的慢作用脱色制剂、至少80％的慢作用脱色制剂、至少90％的慢作用脱色制剂，或至少95％的慢作用脱色制剂。在其他例子中，将样品首先与缓慢作用脱色制剂温育，接着是复染剂。In some instances, samples are incubated simultaneously with a slow-acting destaining agent and a counterstain. In some examples, the concentration of the slow-acting depigmenting formulation in the slow-acting depigmenting formulation/counterstaining formulation combination can range from about 75% to 99%, such as at least 75% of the slow-acting depigmenting formulation, at least 80% of the slow-acting depigmenting formulation , at least 90% slow-acting depigmenting formulation, or at least 95% slow-acting depigmenting formulation. In other examples, samples are first incubated with a slow-acting destaining agent, followed by a counterstain.
本领域技术人员会领会革兰氏染色方法的每个接触步骤在下一个接触步骤之前都可以包括或继之以漂洗步骤。漂洗可以使用能洗掉先前制剂而无不想要的反应的任何随后制剂完成。例如，随后制剂可以是后续的革兰氏染色制剂之一，将其过量与样品接触，并允许排掉，在该情况中将漂洗和使样品与下一个革兰氏染色制剂接触有效组合(例如以图1中显示的次序)。在另一个例子中，在接触步骤之后或作为其一部分加入分别的漂洗步骤(例如图1中显示的那些)。在该例子中，当样品与无活性制剂(例如水或缓冲液)接触时进行漂洗，然后允许排掉。Those skilled in the art will appreciate that each contacting step of the Gram staining method may include or be followed by a rinsing step prior to the next contacting step. Rinsing can be accomplished with any subsequent formulation that washes away the previous formulation without undesired reactions. For example, the subsequent formulation may be one of the subsequent Gram stain formulations, which is contacted in excess with the sample and allowed to drain, in which case an effective combination of rinsing and contacting the sample with the next Gram stain formulation (e.g. in the order shown in Figure 1). In another example, a separate rinse step (such as those shown in Figure 1) is added after or as part of the contacting step. In this example, the rinse is performed while the sample is in contact with the inactive agent (eg, water or buffer) and then allowed to drain.
在一些例子中，样品与慢作用脱色制剂接触或温育的时间长于传统的手动革兰氏染色。例如，使用慢作用脱色制剂需要更长的时间以从革兰氏阴性细胞除去初级染剂-捕获剂(例如CV-I)复合物(及如此初级染剂，例如结晶紫)。在一些例子中，样品与慢作用脱色制剂接触或温育至少30秒、至少45秒、至少60秒、至少120秒、或至少220秒、例如30秒至4分钟，1至5分钟，或2至4分钟。在一些例子中，更长的脱色时间段允许自动化装置完成脱色步骤，如此维持自动化装置在发生超出期望的脱色(如将革兰氏阳性细菌脱色)之前控制脱色步骤完成的能力。如此，在一些例子中，一个或多个步骤之间或之中的时间设计为符合自动化，如通过特定装置部分的革兰氏染色自动化。In some instances, samples are contacted or incubated with slow-acting destaining agents for a longer period of time than traditional manual Gram staining. For example, longer times are required to remove primary stain-capture agent (eg CV-I) complexes (and thus primary stains such as crystal violet) from Gram-negative cells using slow-acting depigmenting formulations. In some examples, the sample is contacted or incubated with the slow-acting depigmenting agent for at least 30 seconds, at least 45 seconds, at least 60 seconds, at least 120 seconds, or at least 220 seconds, such as 30 seconds to 4 minutes, 1 to 5 minutes, or 2 to 4 minutes. In some examples, the longer decolorization time period allows the automated device to complete the decolorization step, thus maintaining the ability of the automated device to control completion of the decolorization step before more than desired decolorization occurs (eg, decolorization of Gram-positive bacteria). Thus, in some instances, the timing between or among one or more steps is designed to allow for automation, such as automation of a Gram stain by a particular device portion.
在一个例子中，将样品与初级染剂、捕获剂或复染剂温育或接触达至少30秒、至少40秒、至少50秒、至少60秒、至少70秒、至少80秒、至少90秒、至少100秒、至少120秒、至少150秒、至少180秒、至少210秒、至少220秒、至少240秒、至少270秒、或至少300秒。另外，在使样品与任意或每种革兰氏染色制剂(初级染剂，捕获剂，缓慢作用脱色制剂和复染剂)接触后，后续的样品与漂洗制剂或与任何随后革兰氏染色制剂的接触在一些例子中可在至少约10秒、20秒、30秒、40秒、50秒、60秒、70秒、80秒、90秒、100秒、120秒、150秒、180秒、210秒、240秒、270秒、或300秒后进行。如此，例如，在使样品与初级染剂接触后，与捕获剂的接触可发生在至少10秒后，如至少30秒或至少1分钟后。在其中包括使用无活性制剂的分开漂洗步骤的例子中，在该漂洗步骤后，与下一个革兰氏染色制剂接触可在至少约0.5秒、1秒、2秒、4秒、6秒、8秒、10秒、20秒、30秒、40秒、50秒、60秒、70秒、80秒、90秒、100秒、120秒、150秒、180秒、210秒、240秒、270秒或300秒后进行。In one example, the sample is incubated or contacted with the primary stain, capture agent or counterstain for at least 30 seconds, at least 40 seconds, at least 50 seconds, at least 60 seconds, at least 70 seconds, at least 80 seconds, at least 90 seconds , at least 100 seconds, at least 120 seconds, at least 150 seconds, at least 180 seconds, at least 210 seconds, at least 220 seconds, at least 240 seconds, at least 270 seconds, or at least 300 seconds. Additionally, after contacting the sample with any or each of the Gram stain formulations (primary stain, capture reagent, slow-acting destain formulation, and counterstain), subsequent samples are treated with the rinse formulation or with any subsequent Gram stain formulation. In some examples, the contact may be at least about 10 seconds, 20 seconds, 30 seconds, 40 seconds, 50 seconds, 60 seconds, 70 seconds, 80 seconds, 90 seconds, 100 seconds, 120 seconds, 150 seconds, 180 seconds, 210 seconds seconds, 240 seconds, 270 seconds, or 300 seconds. Thus, for example, contacting the capture agent may occur at least 10 seconds after contacting the sample with the primary stain, such as at least 30 seconds or at least 1 minute. In instances where a separate rinse step using an inactive formulation is included, contact with the next Gram stain formulation after the rinse step can be at least about 0.5 seconds, 1 second, 2 seconds, 4 seconds, 6 seconds, 8 seconds seconds, 10 seconds, 20 seconds, 30 seconds, 40 seconds, 50 seconds, 60 seconds, 70 seconds, 80 seconds, 90 seconds, 100 seconds, 120 seconds, 150 seconds, 180 seconds, 210 seconds, 240 seconds, 270 seconds or 300 seconds later.
革兰氏染色方法中试剂的使用温度可以是周围环境，或者可以由自动化装置和本地设施来控制，达到对每种制剂相同或对一种或多种制剂有不同的程度。在一个例子中，无活性漂洗制剂比环境空气温度相对要冷。在一些例子中，无活性漂洗制剂在马上与样品接触之前的范围是5°至50℃。在一些例子中，一种或多种革兰氏染色制剂(初级染剂，捕获剂，缓慢作用脱色制剂和复染剂)在马上与样品接触之前为10°至60℃。The temperature at which the reagents used in the Gram staining method can be ambient, or can be controlled by automation and local facilities, to the extent that they are the same for each preparation or different for one or more preparations. In one example, the inactive rinse formulation is relatively cooler than the ambient air temperature. In some instances, the inactive rinse formulation ranges from 5° to 50°C immediately prior to contacting the sample. In some instances, one or more of the Gram stain formulations (primary stain, capture reagent, slow acting destaining formulation, and counterstain) are at 10° to 60° C. immediately prior to contacting the sample.
在一些例子中，调整革兰氏染色制剂的pH，并且可以包括酸、碱和缓冲剂。在一些例子中，一种或多种革兰氏染色制剂(初级染剂，捕获剂，缓慢作用脱色制剂和复染剂)在pH＝3至pH＝8，例如以实现更好的染色和在革兰氏染色后区分细菌细胞和非细菌细胞。In some examples, the pH of the Gram stain formulation is adjusted and may include acids, bases and buffers. In some instances, one or more of the Gram stain formulations (primary stain, capture, slow-acting destain, and counterstain) are at pH=3 to pH=8, e.g., to achieve better staining and in Bacterial and non-bacterial cells were distinguished after Gram staining.
在一些例子中，所述方法还包括检测样品中的革兰氏阳性或革兰氏阴性细菌，例如使用显微镜或其他类似的装置。例如，所述方法可包括确定分析的样品是否含有革兰氏阳性和/或革兰氏阴性细菌。In some examples, the method further includes detecting Gram-positive or Gram-negative bacteria in the sample, for example, using a microscope or other similar device. For example, the method can include determining whether the analyzed sample contains Gram-positive and/or Gram-negative bacteria.
所公开的方法在图1中例示，该图显示了例示性的自动化染色方法，100，其包括革兰氏染色规程101。图1中显示的革兰氏染色规程101具有包括以下的步骤：使样品与初级染剂制剂107接触，使样品与捕获制剂108接触，使样品与缓慢作用脱色制剂109接触，使样品与复染色制剂110接触，和将样品111分类为革兰氏阳性、革兰氏阴性或不确定的。如此，所述方法包括使样品与缓慢作用脱色制剂109接触。图1显示使样品与缓慢作用脱色制剂109接触发生在样品与初级染剂制剂107和捕获制剂108接触之后，但在样品与复染色制剂110接触之前。如上文论述的，每个接触步骤107，108，109，and 110在下一个接触步骤之前可包括或继之以另外的漂洗步骤。例如，在样品与初级染剂制剂107接触之后，可在样品与捕获制剂108接触之前或期间将其漂洗。所述方法还可以包括步骤111，其分类或确定样品中的细菌是革兰氏阴性、革兰氏阳性、或者是否不能进行这类确定。The disclosed method is illustrated in FIG. 1 , which shows an exemplary automated staining method, 100 , including a Gram staining protocol 101 . The Gram staining protocol 101 shown in FIG. 1 has steps including: contacting the sample with a primary stain formulation 107, contacting the sample with a capture formulation 108, contacting the sample with a slow-acting depigmenting formulation 109, contacting the sample with a counterstain Formulation 110 is contacted, and sample 111 is classified as Gram-positive, Gram-negative, or indeterminate. As such, the method includes contacting the sample with a slow acting depigmenting agent 109 . FIG. 1 shows that contacting the sample with the slow-acting destaining formulation 109 occurs after the sample is contacted with the primary stain formulation 107 and the capture formulation 108 , but before the sample is contacted with the counterstain formulation 110 . As discussed above, each contacting step 107, 108, 109, and 110 may include or be followed by an additional rinsing step prior to the next contacting step. For example, after the sample is contacted with the primary stain formulation 107, the sample can be rinsed before or during contact with the capture formulation 108. The method may also include a step 111 of classifying or determining whether the bacteria in the sample are Gram-negative, Gram-positive, or whether such a determination cannot be made.
如此，在样品与任意或每种图1的革兰氏染色制剂接触后(107，108，109和/或110)，后续的与漂洗制剂或与随后的革兰氏染色制剂接触可在至少约10秒、20秒、30秒、40秒、50秒、60秒、70秒、80秒、90秒、100秒、120秒、150秒、180秒、210秒、215秒、220秒、240秒、270秒、或300秒后发生。在其中包括使用无活性制剂的分开漂洗步骤的例子中，在该漂洗步骤后，与图1的下一个革兰氏染色制剂(107、108、109和/或110)接触可在至少约0.5秒、1秒、2秒、4秒、6秒、8秒、10秒、20秒、30秒、40秒、50秒、60秒、70秒、80秒、90秒、100秒、120秒、150秒、180秒、210秒、240秒、270秒、或300秒后进行。Thus, after the sample is contacted with any or each of the Gram stain formulations of FIG. 10 seconds, 20 seconds, 30 seconds, 40 seconds, 50 seconds, 60 seconds, 70 seconds, 80 seconds, 90 seconds, 100 seconds, 120 seconds, 150 seconds, 180 seconds, 210 seconds, 215 seconds, 220 seconds, 240 seconds , 270 seconds, or 300 seconds later. In instances where a separate rinse step using an inactive formulation is included, contact with the next Gram stain formulation (107, 108, 109, and/or 110) of FIG. , 1 second, 2 seconds, 4 seconds, 6 seconds, 8 seconds, 10 seconds, 20 seconds, 30 seconds, 40 seconds, 50 seconds, 60 seconds, 70 seconds, 80 seconds, 90 seconds, 100 seconds, 120 seconds, 150 seconds seconds, 180 seconds, 210 seconds, 240 seconds, 270 seconds, or 300 seconds.
如图1中显示的，在用于自动化革兰氏染色100的方法中，该方法可包括获得样品102和将样品103加载到自动化装置中的步骤。该自动化装置可在平行的105中或在系列的先前104中或在随后的106中、革兰氏染色步骤101中进行测定。先前的104和随后的系列测定106可在相同的样品中进行，但例如在不同的载玻片上进行。先前104和随后系列测定106的例子包括免疫测定和另外的微生物学测定。As shown in Figure 1, in a method for automated Gram staining 100, the method may include the steps of obtaining a sample 102 and loading the sample 103 into an automated device. The automated device can perform the determination in the Gram staining step 101 in parallel 105 or in the previous 104 or in the subsequent 106 of the series. The previous 104 and subsequent series of assays 106 may be performed on the same sample, but eg on different slides. Examples of prior 104 and subsequent series of assays 106 include immunoassays and additional microbiological assays.
自动化automation
在一些例子中，使用自动染色装置进行一个或多个革兰氏染色步骤(例如使样品与初级染剂制剂、捕获剂制剂、慢作用脱色剂制剂、和/或复染色制剂接触)。例如，所公开的方法可与Ventana Medical Systems，Inc.装置如Ventana NexES Special Stainer、Ventana BenchMark Special Stainer、BenchmarkXT、Benchmark Ultra和Discovery系统一起使用。例示性的系统在美国专利第6,352,861号、美国专利第5,654,200号、美国专利第6,582,962号、美国专利第6,296,809号和美国专利第5,595,707号中公开，且关于自动化系统和方法的另外的信息还可见于PCT/US2009/067042。In some examples, one or more Gram staining steps (eg, contacting the sample with a primary stain formulation, a capture agent formulation, a slow-acting destain formulation, and/or a counterstain formulation) are performed using an automated staining device. For example, the disclosed methods can be used with Ventana Medical Systems, Inc. devices such as the Ventana NexES Special Stainer, Ventana BenchMark Special Stainer, BenchmarkXT, Benchmark Ultra, and Discovery systems. Exemplary systems are disclosed in U.S. Patent No. 6,352,861, U.S. Patent No. 5,654,200, U.S. Patent No. 6,582,962, U.S. Patent No. 6,296,809, and U.S. Patent No. 5,595,707, and additional information regarding automated systems and methods can also be found at PCT/US2009/067042.
在一些例子中，用于自动化革兰氏染色的方法是计算机执行的方法，其中算法、自动化装置内的计算机、分开的计算机、和/或分布式计算机系统和/或网络经由一种或多种计算机可读介质中的计算机可执行指令作用于执行任意或所有的革兰氏染色步骤(例如图1中的一个或多个步骤，如图1的步骤107、108、109、110和111)。例如，所公开的革兰氏染色方法的计算机执行(例如如图1中显示的)可有助于样品的追踪、测定法的选择和/或时机和/或测定的步骤、装置的控制、结果的获得和/或分析、数据保留、向医学或其他信息系统的报告和与其交互、决策制定、样品流的变化、和/或通知操作者或其他人员。In some examples, the method for automated Gram staining is a computer-implemented method, wherein an algorithm, a computer within an automated device, a separate computer, and/or a distributed computer system and/or network communicates via one or more The computer-executable instructions in the computer-readable medium are operable to perform any or all of the Gram staining steps (eg, one or more steps in FIG. 1, such as steps 107, 108, 109, 110, and 111 in FIG. 1). For example, computer implementation of the disclosed Gram staining method (eg, as shown in FIG. 1 ) can facilitate tracking of samples, selection and/or timing of assays and/or steps of assays, control of devices, results acquisition and/or analysis, data retention, reporting to and interaction with medical or other information systems, decision making, changes in sample flow, and/or notification to operators or other personnel.
在自动化的革兰氏染色方法中，自动化装置可能需要比传统的手动革兰氏染色方法更多的时间来处理多种样品和进行测试，如以分钟而不是以秒。例如，可就持续时间以及体积和溶剂化强度而言来调整脱色步骤，从而仅从革兰氏阴性细菌适宜地除去初级染剂-捕获剂复合物(例如CV-I复合物)。然而，对于自动化系统如Ventana Medical Systems，Inc.NexES Special Stainer，BenchMark Special Stainer，Benchmark XT，Benchmark Ultra，或Discovery系统，其中分配/施用和漂洗/除去之间的时间固定为确定的最小值，对脱色步骤的控制被限制为改变脱色制剂的体积或溶剂化强度。若体积太小，可发生不一致的脱色，且其可能改变乙醇的溶剂化强度以允许仅从革兰氏阴性细菌中进行初级染剂-捕获剂复合物(例如，CV-I复合物)的选择性脱色。例如，Ventana BenchMark Special Stainer在分配和去除这两个过程之间的明确且不变的固定步骤中运行，且本文显示慢作用脱色制剂，比如包括1，2-丙烷的那些制剂，在该期限内有效地和选择性地使革兰氏阴性细菌脱色。In automated gram staining methods, the automated device may require more time than traditional manual gram staining methods to process multiple samples and perform tests, eg in minutes rather than seconds. For example, the destaining step can be adjusted in terms of duration as well as volume and solvation strength so that only primary stain-capture complexes (eg, CV-I complexes) are desirably removed from Gram-negative bacteria. However, for automated systems such as Ventana Medical Systems, Inc. NexES Special Stainer, BenchMark Special Stainer, Benchmark XT, Benchmark Ultra, or Discovery systems, where the time between dispensing/application and rinsing/removal is fixed at an established minimum, the Control of the depigmentation step is limited to changing the volume or solvation strength of the depigmentation formulation. If the volume is too small, inconsistent decolorization can occur, and it may alter the solvation strength of ethanol to allow selection of primary dye-capture complexes (e.g., CV-I complexes) from Gram-negative bacteria only Sexual depigmentation. For example, the Ventana BenchMark Special Stainer operates in a defined and invariant stationary step between dispensing and removal, and this paper shows that slow-acting depigmenting formulations, such as those including 1,2-propane, Effectively and selectively decolorizes Gram-negative bacteria.
自动化染色设备可部分地通过使用压缩的空气来运行。因此，在一些例子中，当将样品与初级染色制剂、捕获剂制剂、缓慢作用脱色剂制剂和、或复染制剂相接触时，使用压缩的空气将初级染剂制剂、捕获剂制剂、慢作用脱色剂制剂，和、或复染剂递送至样品。Automated dyeing equipment can be operated in part by using compressed air. Thus, in some instances, the primary stain preparation, capture agent preparation, slow-acting A depigmentation agent preparation, and/or a counterstain is delivered to the sample.
在一些例子中，所述方法包括将待分析有无细菌的样品加载到自动化染色的设备中。In some examples, the method includes loading a sample to be analyzed for bacteria into an automated staining device.
在一个特定的例子中，提供了用于利用自动化染色设备在载玻片上对细菌进行革兰氏染色的方法。自动化染色设备可包括用于持有多个载玻片的转盘式传送工具，其中每个载玻片在其上具有待染色的样品，比如已知含有或怀疑含有细菌的样品。自动化染色设备还可包括用于以预定的速度旋转该转盘式传送工具的工具和在转盘式传送工具的旋转过程中用于导引和控制试剂(比如革兰氏染色试剂或其他染剂)在载玻片和样品上的应用的装置。该方法可包括将初级染剂试剂(比如含有结晶紫的一种试剂)导引到转盘式传送工具中的载玻片上以将载玻片上的细菌与初级染剂试剂相接触；将水、水-表面活性剂混合物，或缓冲液导引到转盘式传送工具中的载玻片上以从载玻片冲洗初级染剂试剂，所述冲洗例如在允许多余的染剂去除的条件下进行；将捕获试剂(比如含有碘的一种试剂)导引到转盘式传送工具中的载玻片上以将载玻片上的细菌与捕获试剂相接触，所述接触例如在允许在样品中的格兰仕阳性细菌中形成初级染剂-捕获剂复合物的条件下进行，将水或其他缓冲液导引到转盘式传送工具中的载玻片上以从载玻片冲掉过量的捕获试剂；将慢作用脱色制剂和复染剂单独地或共同地导引到转盘式传送工具中的载玻片上，所述导引例如在允许去除样品中的革兰氏阴性细菌中的初级染剂的条件下进行；和将水和缓冲液导引到转盘式传送工具中的载玻片上以从载玻片冲洗过量的慢作用脱色制剂和/或复染剂(若慢作用脱色制剂和复染剂是分别添加的，该方法可包括期间的洗涤步骤)。方法还可包括在载玻片上检测革兰氏阳性或革兰氏阴性细菌。In a specific example, methods are provided for Gram staining bacteria on glass slides using automated staining equipment. Automated staining equipment may include a carousel for holding a plurality of slides, each slide having thereon a sample to be stained, such as a sample known to contain or suspected to contain bacteria. The automated staining apparatus may also include means for rotating the carousel at a predetermined speed and for guiding and controlling the flow of reagents (such as Gram stains or other dyes) during the rotation of the carousel. Device for application on slides and samples. The method may include directing a primary stain reagent, such as one containing crystal violet, onto a glass slide in a carousel to contact the bacteria on the slide with the primary stain reagent; - a surfactant mixture, or buffer, is directed onto the slide in the carousel to rinse the primary stain reagent from the slide, for example under conditions that allow excess stain to be removed; capture A reagent, such as one containing iodine, is directed onto the slide in the carousel to bring the bacteria on the slide into contact with the capture reagent, such as in allowing Galanz-positive bacteria in the sample to form Under conditions of the primary stain-capture complex, water or other buffer is directed onto the slide in a carousel to flush excess capture reagent from the slide; slow-acting destaining preparation and complex Stains are introduced individually or collectively onto the glass slide in the carousel, for example under conditions that allow removal of the primary stain from Gram-negative bacteria in the sample; and water and The buffer is directed onto the slides in the carousel to flush excess slow-acting destaining preparation and/or counterstain from the slides (this method can be used if the slow-acting destaining preparation and counterstain are added separately). including washing steps in between). The method may also include detecting Gram-positive or Gram-negative bacteria on the slide.
慢作用脱色制剂slow acting depigmentation preparation
所公开的方法中使用的慢作用脱色制剂依赖于慢作用脱色剂如1，2-丙二醇和乙二醇的鉴定。本文显示了脱色剂1，2-丙二醇，或通常所称的丙二醇(PG)，可在自动化的染色仪的定时的固定步骤(4分钟)内有效地从革兰氏阴性细菌中脱色CV-I复合物，所述染色仪例如BenchMark Special Stainer。与其他脱色剂相比，PG较慢地溶解革兰氏阴性细菌的脂多糖并允许CV-I复合物从细菌细胞膜(肽聚糖)扩散，从而仅允许革兰氏阴性细胞的选择性脱色以较慢的速率发生。因此PG允许通过自动化的染色系统进行革兰氏染色，所述自动化的染色系统在脱色剂应用和去除之间具有设定的时间，该设定的时间基本上长于利用常规脱色剂所用的时间。The slow-acting depigmenting formulations used in the disclosed methods rely on the identification of slow-acting depigmenting agents such as 1,2-propanediol and ethylene glycol. Here it is shown that the depigmenting agent 1,2-propanediol, or commonly known as propylene glycol (PG), can efficiently decolorize CV-I from Gram-negative bacteria within a timed fixation step (4 minutes) in an automated stainer Composite, said stainer such as BenchMark Special Stainer. Compared to other depigmenting agents, PG dissolves the lipopolysaccharide of Gram-negative bacteria more slowly and allows the CV-I complex to diffuse from the bacterial cell membrane (peptidoglycan), thereby allowing selective depigmentation of only Gram-negative cells to slower rate occurs. PG thus allows Gram staining by an automated staining system with a set time between depigmentation application and removal that is substantially longer than that used with conventional depigmentation agents.
这些和任何其他慢作用脱色剂中的一种或多种可以以纯的形式、用水或缓冲液稀释的形式、与快作用剂一起的形式来使用以产生具有所需脱色速率的脱色制剂。在一个例子中，慢作用脱色制剂包括慢作用脱色剂，如1，2-丙二醇，乙二醇，或其组合。在一个具体例子中，慢作用脱色制剂是1，2-丙二醇或乙二醇或由它们组成。在一些例子中，水可包括在慢作用脱色制剂中，例如以范围从约0％至25％，如0.5％至25％，1％至20％，5％至10％，如约10％的浓度。One or more of these and any other slow-acting depigmenting agents may be used neat, diluted with water or buffer, together with fast-acting agents to produce a depigmenting preparation having the desired depigmentation rate. In one example, the slow-acting depigmenting formulation includes a slow-acting depigmenting agent, such as 1,2-propanediol, ethylene glycol, or combinations thereof. In a specific example, the slow-acting depigmenting agent is or consists of 1,2-propanediol or ethylene glycol. In some examples, water may be included in the slow-acting depigmentation formulation, e.g., at a concentration ranging from about 0% to 25%, such as 0.5% to 25%, 1% to 20%, 5% to 10%, such as about 10%. .
在一些例子中，慢作用脱色制剂可包括缓慢作用脱色剂和快速作用脱色剂的混合物，如至少80％、至少81％、至少82％、至少83％、至少84％、至少85％、至少86％、至少87％、至少88％、至少89％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或至少99.9％的缓慢作用脱色剂(例如1，2-丙二醇，乙二醇或其组合)，如80％至99.99％、80％至95％、90％至99或90％至95％的缓慢作用脱色剂，和不超过20％、不超过19％、不超过18％、不超过17％、不超过16％、不超过15％、不超过14％、不超过13％、不超过12％、不超过11％、不超过10％、不超过9％、不超过8％、不超过7％、不超过6％、不超过5％、不超过4％、不超过3％、不超过2％、不超过1％、不超过0.5％或不超过0.01％的快速作用脱色剂，如0.01％至20％、1％至20％、1％至10％或1％至5％的快速作用脱色剂。快速作用脱色剂的例子包括丙二醇单甲醚、二乙醚、乙醇、丙酮、甲醇、1-丙醇、2-丙醇、1-丁醇或其组合，如乙醇和丙酮的共混物和乙醇和其他醇类如甲醇或2-丙醇的共混物。In some examples, the slow-acting depigmenting formulation may comprise a mixture of slow-acting depigmenting agents and fast-acting depigmenting agents, such as at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86% %, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, At least 99% or at least 99.9% of a slow-acting depigmenting agent (such as 1,2-propanediol, ethylene glycol, or combinations thereof), such as 80% to 99.99%, 80% to 95%, 90% to 99, or 90% to 95% % slow acting depigmenting agents, and not more than 20%, not more than 19%, not more than 18%, not more than 17%, not more than 16%, not more than 15%, not more than 14%, not more than 13%, not more than 12%, not exceeding 11%, not exceeding 10%, not exceeding 9%, not exceeding 8%, not exceeding 7%, not exceeding 6%, not exceeding 5%, not exceeding 4%, not exceeding 3%, not exceeding 2%, not more than 1%, not more than 0.5%, or not more than 0.01% fast acting depigmentation agents, such as 0.01% to 20%, 1% to 20%, 1% to 10%, or 1% to 5% fast acting Depigmentation agent. Examples of fast acting depigmenting agents include propylene glycol monomethyl ether, diethyl ether, ethanol, acetone, methanol, 1-propanol, 2-propanol, 1-butanol or combinations thereof, such as blends of ethanol and acetone and ethanol and Blends of other alcohols such as methanol or 2-propanol.
样品sample
样品可以是待分析细菌的存在的任何材料。样品可以是生物的或非生物的且可获自受试者、环境、系统或过程。因此，在一些例子中，方法包括获得样品。例如，样品可获自已知有或怀疑有细菌感染的受试者，或获自已知或怀疑被细菌污染的来源。The sample can be any material to be analyzed for the presence of bacteria. A sample can be biological or non-biological and can be obtained from a subject, environment, system or process. Thus, in some instances, methods include obtaining a sample. For example, a sample can be obtained from a subject known or suspected to have a bacterial infection, or from a source known or suspected to be contaminated with bacteria.
在一个例子中，样品获自受试者，比如人类受试者。该样品可以是固体的或流体的样品，获自受试者或由受试者排泄或分泌，所述受试者比如细胞、细胞裂解物、外周血(或其成分，比如血清或血浆)、尿、胆汁、腹水、唾液、颊刮拭物、组织活检物(比如肿瘤活检物或淋巴结活检物)、外科样本、骨髓、羊水样品、细针吸入物、子宫颈样品(例如，PAP涂片、来自外子宫颈或内子宫颈的细胞)、脑脊髓液、水状体或玻璃体、渗出液、流出物(例如，获自脓肿或感染或炎症的任何其他位点的流体)、获自关节(例如，正常的关节或患病的关节，比如类风湿性关节炎、骨关节炎、痛风关节炎或败血关节炎)的流体和尸检物质。In one example, a sample is obtained from a subject, such as a human subject. The sample may be a solid or fluid sample obtained from or excreted or secreted by a subject, such as cells, cell lysates, peripheral blood (or components thereof, such as serum or plasma), Urine, bile, ascites, saliva, buccal swab, tissue biopsy (eg, tumor biopsy or lymph node biopsy), surgical specimen, bone marrow, amniotic fluid sample, fine needle aspiration, cervical sample (eg, PAP smear, cells from the ectocervix or endocervix), cerebrospinal fluid, aqueous humor or vitreous, exudate, effusion (for example, fluid obtained from an abscess or any other site of infection or inflammation), obtained from a joint ( For example, normal joints or diseased joints, such as rheumatoid arthritis, osteoarthritis, gouty arthritis, or septic arthritis) fluid and autopsy material.
在一个例子中，样品获自环境，比如获自水体、空气、沉淀物、灰尘、土壤或获自自然环境或居民环境、商业、工业、医药、学校、农业或其他人造环境(例如，食物加工、生产和消耗设施和处理环境)中，且可获自工业来源，比如农场、废物流或水源。因此，样品通常可以是获自已知含有细菌、微生物或多细胞物质的那些。In one example, the sample is obtained from the environment, such as from a body of water, air, sediment, dust, soil, or from a natural or residential, commercial, industrial, pharmaceutical, school, agricultural or other man-made environment (e.g., food processing , production and consumption facilities and disposal environments) and can be obtained from industrial sources such as farms, waste streams or water sources. Thus, samples may generally be those obtained from those known to contain bacteria, microorganisms or multicellular material.
在一个例子中，样品是食物样品(比如肉、水果、奶制品或蔬菜样品)活活子食物加工植物的样品。In one example, the sample is a sample of a food sample (such as a meat, fruit, dairy or vegetable sample) or a sample of a live food processing plant.
在一些例子中，样品是收集的流体、刮擦物、过滤物或培养物。在一个例子中，样品是细胞学样品。在具体的例子中，直接使用样品(例如新鲜的或冷冻的)，或在使用前处理样品，例如通过浓缩、稀释、固定(例如使用福尔马林或热)和/或包埋于蜡中(如福尔马林固定石蜡包埋(FFPE)样品)来进行处理。在一个例子中，样品是热固定于显微镜载玻片的。In some examples, the sample is a collected fluid, scraping, filter, or culture. In one example, the sample is a cytology sample. In specific examples, the sample is used directly (e.g. fresh or frozen), or the sample is treated prior to use, e.g. by concentrating, diluting, fixing (e.g. using formalin or heat) and/or embedding in wax (such as formalin-fixed paraffin-embedded (FFPE) samples) for processing. In one example, the sample is heat fixed to a microscope slide.
另外的测定additional determination
在一些例子中，公开的方法可包括对样品进行另外的测定，例如以确定或鉴定样品中的特定的细菌。另外的测定可在革兰氏染色之前或之后，或甚至于革兰氏染色同时或同期进行。例如，可将样品涂覆于多个载玻片(例如，不同的载玻片或FFPE样品的不同的切片)，其中对每个载玻片进行特定的测定。因此，可对相同的样品载玻片和/或取自相同样品并固定在不同的载玻片上的多个切片，和/或固定在相同的和/或不同的载玻片上的来自相同的和/或不同的受试者的不同的样品平行地和/或连续地进行多个测定。In some examples, the disclosed methods can include performing additional assays on the sample, for example, to identify or identify specific bacteria in the sample. Additional assays can be performed before or after Gram staining, or even simultaneously or concurrently with Gram staining. For example, a sample can be applied to multiple slides (eg, different slides or different sections of an FFPE sample), with specific assays performed on each slide. Thus, multiple sections of the same sample slide and/or taken from the same sample and mounted on different slides, and/or slices from the same and/or different slides mounted on the same and/or different Multiple assays are performed in parallel and/or serially on different samples of different subjects and/or different subjects.
在一个例子中，对样品进行免疫测定，例如通过与对特定的靶细菌具有特异性的一种或多种抗体孵育，并利用标记物(比如抗体上的标记物或通过使用标记的第二抗体)检测来进行免疫测定。示例性的可检测的标记物包括荧光团、半抗原、酶、放射性标记物、量子点和本领域中已知的其他可检测标记物。In one example, the sample is subjected to an immunoassay, e.g., by incubation with one or more antibodies specific for a particular target bacterium, and utilizing a label (such as a label on the antibody or by using a labeled secondary antibody ) detection for immunoassay. Exemplary detectable labels include fluorophores, haptens, enzymes, radiolabels, quantum dots, and others known in the art.
在另一个例子中，对样品进行另外的微生物测定。例如，可将样品与一种或多种其他染料相接触，比如一下染料中的一种或多种：Acid Fast Bacilli(AFB)III，Alcian Blue，Alcian Blue for Periodic Acid Schiff(PAS)，AlcianYellow，Congo Red，Diastase，Elastic，Giemsa，Grocott′s Methenamine Silverstain(GMS)II，iron，Jones Light Green，Jones，Light Green for PAS，Mucicamine PAS，Reticulum，Steiner II，Trichrome Blue和Trichrome Green。在一个例子中，将样品与Ziehl-Neelsen或相似的染料相接触，例如以检测显示耐酸性但有时难以利用革兰氏染色鉴定的分支杆菌或诺卡氏菌。In another example, additional microbiological assays are performed on the sample. For example, the sample can be contacted with one or more other dyes, such as one or more of the following: Acid Fast Bacilli (AFB) III, Alcian Blue, Alcian Blue for Periodic Acid Schiff (PAS), Alcian Yellow, Congo Red, Diastase, Elastic, Giemsa, Grocott's Methenamine Silverstain (GMS) II, iron, Jones Light Green, Jones, Light Green for PAS, Mucicamine PAS, Reticulum, Steiner II, Trichrome Blue and Trichrome Green. In one example, the sample is exposed to a Ziehl-Neelsen or similar dye, eg, to detect Mycobacteria or Nocardia that show acid resistance but are sometimes difficult to identify using Gram stains.
试剂盒Reagent test kit
提供了可与公开的方法一起使用的试剂盒。在一些例子中，该试剂盒可与具有较长的脱色步骤(如花费至少30秒、至少1分钟、至少2分钟、至少3分钟、至少4分钟或至少5分钟来完成的脱色步骤)的自动化的革兰氏染色方法一起使用。Kits are provided that can be used with the disclosed methods. In some examples, the kit can be automated with a longer destaining step (e.g., a destaining step that takes at least 30 seconds, at least 1 minute, at least 2 minutes, at least 3 minutes, at least 4 minutes, or at least 5 minutes to complete). used with the Gram staining method.
在具体的例子中，公开的试剂盒包括容器中(例如，小瓶或器皿)的缓慢作用脱色制剂。在一些例子中，试剂盒还包括用于进行革兰氏染色方法的其他步骤中的一项或多项的试剂。例如，试剂盒还可包括初级染剂制剂、捕获剂制剂和/或复染制剂，例如其中每个制剂在分开的容器中。In specific examples, the disclosed kits include a slow-acting depigmenting formulation in a container (eg, vial or vessel). In some instances, the kit also includes reagents for performing one or more of the other steps of the Gram stain method. For example, a kit may also include a primary stain formulation, a capture agent formulation, and/or a counterstain formulation, eg, wherein each formulation is in a separate container.
在一个例子中，慢作用脱色制剂的容器包括一种或多种慢作用脱色剂，如1，2-丙二醇、乙二醇或其组合。在一个特定的例子中，慢作用脱色制剂1，2-丙二醇或乙二醇或由该二者组成。在一些例子中，慢作用脱色制剂可包括慢作用脱色剂和快作用脱色剂的组合，如至少80％、至少81％、至少82％、至少83％、至少84％、至少85％、至少86％、至少87％、至少88％、至少89％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或至少99.9％的一种或多种慢作用脱色剂(例如1，2-丙二醇、乙二醇或其组合)、如80％至99.99％、80％至99％、80％至90％、90％至99.9％或90％至98％的慢作用脱色剂，和不超过20％、不超过19％、不超过18％、不超过17％、不超过16％、不超过15％、不超过14％、不超过13％、不超过12％、不超过11％、不超过10％、不超过9％、不超过8％、不超过7％、不超过6％、不超过5％、不超过4％、不超过3％、不超过2％、不超过1％、不超过0.5％或不超过0.01％的一种或多种快作用脱色剂(例如丙二醇单甲醚、二乙醚、乙醇、丙酮、甲醇、1-丙醇、2-丙醇、1-丁醇，或其组合，如乙醇和丙酮的共混物和乙醇和其他醇类如甲醇或2-丙醇的共混物)，如0.01％至20％、0.1％至20％、0.1％至10％、1％至20％或1％至10％的快作用脱色剂。In one example, the container of slow-acting depigmenting formulation includes one or more slow-acting depigmenting agents, such as 1,2-propanediol, ethylene glycol, or combinations thereof. In a specific example, the slow-acting depigmenting agent is 1,2-propanediol or ethylene glycol or consists of both. In some examples, the slow-acting depigmenting formulation can comprise a combination of slow-acting depigmenting agents and fast-acting depigmenting agents, such as at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86% %, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, At least 99% or at least 99.9% of one or more slow-acting depigmenting agents (such as 1,2-propanediol, ethylene glycol or combinations thereof), such as 80% to 99.99%, 80% to 99%, 80% to 90% %, 90% to 99.9%, or 90% to 98% of slow-acting depigmenting agents, and not more than 20%, not more than 19%, not more than 18%, not more than 17%, not more than 16%, not more than 15%, Not more than 14%, not more than 13%, not more than 12%, not more than 11%, not more than 10%, not more than 9%, not more than 8%, not more than 7%, not more than 6%, not more than 5%, Not more than 4%, not more than 3%, not more than 2%, not more than 1%, not more than 0.5%, or not more than 0.01% of one or more fast-acting depigmenting agents (such as propylene glycol monomethyl ether, diethyl ether, ethanol , acetone, methanol, 1-propanol, 2-propanol, 1-butanol, or combinations thereof such as blends of ethanol and acetone and blends of ethanol and other alcohols such as methanol or 2-propanol) , such as 0.01% to 20%, 0.1% to 20%, 0.1% to 10%, 1% to 20%, or 1% to 10% fast-acting depigmentation agent.
在一个例子中，初级染剂制剂的容器包括结晶紫(CV)或甲紫。在一个例子中，初级染剂制剂包括结晶紫，浓度至少0.5g/L、至少0.75g/L、至少1g/L、至少2g/L、至少3g/L、至少4g/L或至少5g/L、如1-4g/L，例如3/L。在一些例子中，CV存在于含有异丙醇、乙醇、甲醇和水的溶液中，如50ml异丙醇、50ml乙醇、甲醇和900ml水。在一些例子中，CV或甲紫溶液含有草酸铵以增强稳定性。In one example, the container of the primary stain formulation includes crystal violet (CV) or methyl violet. In one example, the primary dye formulation comprises crystal violet at a concentration of at least 0.5 g/L, at least 0.75 g/L, at least 1 g/L, at least 2 g/L, at least 3 g/L, at least 4 g/L, or at least 5 g/L , such as 1-4g/L, for example 3/L. In some examples, the CV is present in a solution containing isopropanol, ethanol, methanol, and water, such as 50 ml isopropanol, 50 ml ethanol, methanol, and 900 ml water. In some instances, the CV or Violet solution contains ammonium oxalate to enhance stability.
在一个例子中，捕获剂制剂的容器包括碘，如革兰氏碘或聚乙烯吡咯烷酮-碘(PVP-碘)溶液。在一些例子中，革兰氏碘包括至少0.1％碘(例如至少0.2％、至少0.5％或至少1％，如约0.1％-1％碘)和至少0.1％碘化钾(例如至少0.2％、至少0.5％、至少1％、至少1.5％，或至少2％，如约0.1％-2％碘化钾)，例如于水和乙醇混合物中。在一些例子中，PVP-碘可包括至少1％聚乙烯吡咯烷酮(例如至少2％、至少5％、至少10％、至少20％、至少30％，或至少35％，如约1-35％聚乙烯吡咯烷酮)和水中的至少1％碘(例如至少2％、至少5％、至少10％、至少20％、至少30％，或至少35％，如约1％-35％碘)(例如参见美国专利第2,739,922号和第3,898,326号)。In one example, the container of the capture agent formulation includes iodine, such as Gram's iodine or polyvinylpyrrolidone-iodine (PVP-iodine) solution. In some examples, Gram iodine comprises at least 0.1% iodine (e.g., at least 0.2%, at least 0.5%, or at least 1%, such as about 0.1%-1% iodine) and at least 0.1% potassium iodide (e.g., at least 0.2%, at least 0.5% , at least 1%, at least 1.5%, or at least 2%, such as about 0.1%-2% potassium iodide), for example in a water and ethanol mixture. In some examples, PVP-iodine can include at least 1% polyvinylpyrrolidone (e.g., at least 2%, at least 5%, at least 10%, at least 20%, at least 30%, or at least 35%, such as about 1-35% polyethylene pyrrolidone) and at least 1% iodine in water (eg, at least 2%, at least 5%, at least 10%, at least 20%, at least 30%, or at least 35%, such as about 1%-35% iodine) (see, for example, U.S. Patent No. 2,739,922 and 3,898,326).
在一个例子中，复染制剂的容器包括洋红(如碳酸洋红或碱性洋红)、中性红和/或番红O。在一个例子中，含水复染制剂包括至少0.01％、至少0.02％、至少0.03％、至少0.05％、至少0.1％、至少0.2％、至少0.3％、至少0.4％，或至少0.5％，如从0.02％至0.50％的浓度的洋红。在一个例子中，复染制剂包括至少0.01％、至少0.02％、至少0.03％、至少0.05％、至少0.1％、至少0.2％、至少0.3％、至少0.4％，或至少0.5％，如从0.02％至1％的浓度的中性红。在一个例子中，复染制剂包括至少0.1％，如至少0.2％、至少0.3％、至少0.4％、至少0.5％、至少0.6％、至少0.7％，或至少0.8％，如0.1％至1％或0.1％至0.8％浓度的番红O。可在水中配制复染剂，但在一些例子中，复染剂可包括1％-20％乙醇或甲醇。In one example, the container of counterstain formulation includes a carmine (such as carboxy carbamate or basic carmine), neutral red and/or safranin O. In one example, the aqueous counterstain formulation comprises at least 0.01%, at least 0.02%, at least 0.03%, at least 0.05%, at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, or at least 0.5%, such as from 0.02 % to 0.50% concentration of magenta. In one example, the counterstain formulation comprises at least 0.01%, at least 0.02%, at least 0.03%, at least 0.05%, at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, or at least 0.5%, such as from 0.02% Neutral red to a concentration of 1%. In one example, the counterstain formulation comprises at least 0.1%, such as at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, or at least 0.8%, such as from 0.1% to 1% or Safranin O at a concentration of 0.1% to 0.8%. Counterstains can be formulated in water, but in some instances, counterstains can include 1%-20% ethanol or methanol.
在一些例子中，容器或器皿包括初级染剂制剂、捕获剂制剂、缓慢作用脱色剂制剂，和/或复染剂制剂，所述容器或器皿经配置或成形以用于装载入自动的染色设备中。例如，可将容器配置为与Ventana Medical Systems，Inc.设备一起使用，如NexES Special Stainer或Ventana BenchMark Special Stainer。在一些例子中，可将容器配置为与Benchmark XT，Benchmark Ultra或Discovery系统一起使用，如美国专利第6,352,861号、美国专利第5,654,200号、美国专利第6,582,962号、美国专利第6,296,809号和美国专利第5,595,707号中所公开的那些，其均通过引用并入本文。关于自动化的系统和方法的另外的信息还可见于PCT/US2009/067042，其通过引用并入本文。在一个例子中，容器是化学抗性的聚丙烯/聚乙烯小瓶，如Ventana BenchMark分药器。In some examples, a container or vessel includes a primary stain formulation, a capture agent formulation, a slow-acting destain formulation, and/or a counterstain formulation configured or shaped for loading into an automated staining agent. in the device. For example, containers can be configured for use with Ventana Medical Systems, Inc. equipment, such as the NexES Special Stainer or the Ventana BenchMark Special Stainer. In some examples, containers can be configured for use with Benchmark XT, Benchmark Ultra, or Discovery systems, such as U.S. Patent No. 6,352,861, U.S. Patent No. 5,654,200, U.S. Patent No. 6,582,962, U.S. Patent No. No. 5,595,707, which are all incorporated herein by reference. Additional information on automated systems and methods can also be found in PCT/US2009/067042, which is incorporated herein by reference. In one example, the container is a chemically resistant polypropylene/polyethylene vial, such as a Ventana BenchMark dispenser.
试剂盒还可包括一个或多个显微镜载玻片，如适宜于固定的载玻片和在一些例子中为固定样品的载玻片，以及盖玻片、吸液器或其组合。Kits may also include one or more microscope slides, such as slides suitable for mounting and, in some instances, sample mounting, as well as coverslips, pipettes, or combinations thereof.
任选地，试剂盒还可包括对照载玻片以用于验证革兰氏染色方案的试剂和适当的执行。例如，一组对照载玻片可包括革兰氏阳性细菌，如金黄色葡萄球菌(或本文所列的那些中的任何细菌)，且另一组对照载玻片可包括革兰氏阴性细菌，如大肠杆菌(或本文所列的那些中的任何细菌)。在一些例子中，试剂盒中的对照载玻片不包括细菌。Optionally, the kit may also include control slides for use in verifying reagents and proper execution of the Gram staining protocol. For example, one set of control slides can include Gram-positive bacteria, such as Staphylococcus aureus (or any of those listed herein), and another set of control slides can include Gram-negative bacteria, Such as E. coli (or any bacteria among those listed herein). In some instances, control slides in the kit do not include bacteria.
任选地，试剂盒还可包括用于进行另外的测定的另外的试剂。例如，试剂盒可包括包含其他染料或染剂的一个或多个器皿或容器，如包括以下染剂中的一种或多种的容器：Acid Fast Bacilli(AFB)III、Alcian Blue、Alcian Bluefor Periodic Acid Schiff(PAS)、Alcian Yellow、Congo Red、Diastase、Elastic、Giemsa、Grocott′s Methenamine Silver stain(GMS)II、iron、Jones Light Green、Jones、Light Green for PAS、Mucicamine PAS、Reticulum、Steiner II、TrichromeBlue和Trichrome Green。在一个例子中，试剂盒可包括包含细菌特异性的抗体的器皿或容器。在一些例子中，试剂盒包括包含经标记的第二抗体的器皿或容器(例如用荧光团或量子点标记的)。Optionally, the kit may also include additional reagents for performing additional assays. For example, the kit may include one or more vessels or containers containing other dyes or stains, such as containers containing one or more of the following stains: Acid Fast Bacilli (AFB) III, Alcian Blue, Alcian Blue for Periodic Acid Schiff (PAS), Alcian Yellow, Congo Red, Diastase, Elastic, Giemsa, Grocott's Methenamine Silver stain (GMS) II, iron, Jones Light Green, Jones, Light Green for PAS, Mucicamine PAS, Reticulum, Steiner II, Trichrome Blue and Trichrome Green. In one example, a kit can include a vessel or container comprising an antibody specific for a bacterium. In some examples, the kit includes a vessel or container comprising a labeled second antibody (eg, labeled with a fluorophore or quantum dot).
慢作用脱色组合物slow acting depigmenting composition
公开内容还提供了可用于本文提供的试剂盒和方法的慢作用脱色制剂。在一个例子中，慢作用脱色制剂包括慢作用脱色剂和快作用脱色剂的混合物，如一种或多种慢作用脱色剂和一种或多种快作用脱色剂。The disclosure also provides slow-acting depigmenting formulations useful in the kits and methods provided herein. In one example, the slow-acting depigmenting formulation includes a mixture of slow-acting depigmenting agents and fast-acting depigmenting agents, such as one or more slow-acting depigmenting agents and one or more fast-acting depigmenting agents.
在一个例子中，慢作用脱色制剂包括一种或多种慢作用脱色剂，如至少80％、至少81％、至少82％、至少83％、至少84％、至少85％、至少86％、至少87％、至少88％、至少89％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％，或至少99.9％的一种或多种慢作用脱色剂(例如1，2-丙二醇，乙二醇或其组合)，如80％至99.99％的一种或多种慢作用脱色剂。在一个例子中，慢作用脱色制剂包括一种或多种快作用脱色剂，如不超过20％、不超过19％、不超过18％、不超过17％、不超过16％、不超过15％、不超过14％、不超过13％、不超过12％、不超过11％、不超过10％、不超过9％、不超过8％、不超过7％、不超过6％、不超过5％、不超过4％、不超过3％、不超过2％、不超过1％、不超过0.5％或不超过0.01％的一种或多种快作用脱色剂(例如丙二醇单甲醚，二乙醚，乙醇，丙酮、甲醇、1-丙醇，2-丙醇，1-丁醇或其组合，如乙醇和丙酮的共混物和乙醇和其他醇类如甲醇或2-丙醇的共混物)，如0.01％至20％，0.1％至20％，0.1％至10％，1％至20％，或1％至10％的快速作用脱色剂。In one example, the slow-acting depigmenting formulation comprises one or more slow-acting depigmenting agents, such as at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% , or at least 99.9% of one or more slow-acting depigmenting agents (eg, 1,2-propanediol, ethylene glycol, or combinations thereof), such as 80% to 99.99% of one or more slow-acting depigmenting agents. In one example, the slow-acting depigmenting formulation includes one or more fast-acting depigmenting agents, such as not more than 20%, not more than 19%, not more than 18%, not more than 17%, not more than 16%, not more than 15% , not exceeding 14%, not exceeding 13%, not exceeding 12%, not exceeding 11%, not exceeding 10%, not exceeding 9%, not exceeding 8%, not exceeding 7%, not exceeding 6%, not exceeding 5% , not more than 4%, not more than 3%, not more than 2%, not more than 1%, not more than 0.5% or not more than 0.01% of one or more fast-acting decolorizing agents (such as propylene glycol monomethyl ether, diethyl ether, ethanol, acetone, methanol, 1-propanol, 2-propanol, 1-butanol or combinations thereof such as blends of ethanol and acetone and blends of ethanol and other alcohols such as methanol or 2-propanol) , such as 0.01% to 20%, 0.1% to 20%, 0.1% to 10%, 1% to 20%, or 1% to 10% fast acting depigmentation agent.
实施例1Example 1
革兰氏染色脱色剂的比较Comparison of Gram Stain Depigmentation Agents
该实施例描述了用于在革兰氏染色中比较脱色剂以优化脱色的速率的方法。根据Carson F，H1adik C.Histotechnology：A Self Instructional Text，第3版，第231-233页.Hong Kong：American Society for Clinical Pathology Press；2009(作为参考并入本文)制备了结晶紫和革兰氏碘。This example describes a method for comparing depigmenting agents in Gram staining to optimize the rate of depigmentation. Crystal Violet and Gram were prepared according to Carson F, Hiadik C. Histotechnology: A Self Instructional Text, 3rd Edition, pp. 231-233. Hong Kong: American Society for Clinical Pathology Press; 2009 (incorporated herein by reference). iodine.
利用标准的组织学技术对石蜡包埋的革兰氏阳性对照的组织切片进行脱蜡。然后在自动化的原型染色仪上对这些切片进行染色，其中将结晶紫逐滴(～90μL每滴)分散在水平放置的组织切片上，使其孵育持续约4分钟，且然后利用水性表面活性剂溶液冲洗。然后将革兰氏碘逐滴涂覆至切片，且将其孵育持续约4分钟，然后进行冲洗。此时，利用带刻度的移液器人工地将脱色溶剂添加至组织切片，然后以指定的时间段利用水性表面活性剂溶液人工地冲洗。然后人工地涂覆Carbol Fuchsin和Tartrazine的复染剂(分别为90秒和15秒孵育)。然后对载玻片进行脱水并进行二甲苯透明，然后利用永久固定介质覆盖盖玻片。在显微镜(20-200x)下检查，从而评估溶剂对于使革兰氏(-)细菌脱色的选择性。结果显示于下表中。Paraffin-embedded Gram-positive control tissue sections were deparaffinized using standard histological techniques. These sections were then stained on an automated prototype stainer in which crystal violet was dispensed dropwise (~90 μL per drop) over horizontal tissue sections, incubated for approximately 4 minutes, and then treated with aqueous surfactant Solution rinse. Gram's iodine was then applied dropwise to the sections and incubated for approximately 4 minutes before being rinsed. At this point, destaining solvent was manually added to the tissue sections using a graduated pipette, followed by manual rinsing with an aqueous surfactant solution for a specified period of time. Carbol Fuchsin and Tartrazine counterstains were then manually applied (90 sec and 15 sec incubations, respectively). Slides were then dehydrated and cleared in xylene, and coverslipped with permanent mounting medium. Examine under a microscope (20-20Ox) to assess the selectivity of the solvent for decolorizing Gram(-) bacteria. The results are shown in the table below.
*+＝革兰氏(-)为红色，革兰氏(+)为蓝色；*+ = Gram (-) is red, Gram (+) is blue;
0＝革兰氏(+)的最少脱色；0 = minimal depigmentation of Gram (+);
-＝脱色的革兰氏(+)细菌- = depigmented Gram (+) bacteria
为展示可能应用本公开内容的原理的许多可能的实施方案，应认识到所示出的实施方案仅是公开内容的例子，且不应理解为限制本发明的范围。反之，本公开内容的范围由以下的权利要求所限定。因此我们要求我们的发明在这些权利要求的范围和精神中的所有权益。In order to demonstrate the many possible embodiments to which the principles of the disclosure may be applied, it should be recognized that the illustrated embodiments are only examples of the disclosure and should not be taken as limiting the scope of the invention. Rather, the scope of the present disclosure is defined by the following claims. We therefore claim all rights to our invention within the scope and spirit of these claims.
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810139303.XACN108181155B (en) | 2012-03-19 | 2013-02-22 | Gram staining method with improved decolorization of crystal violet-iodine complex of gram negative bacteria |
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US201261612520P | 2012-03-19 | 2012-03-19 | |
| US61/612,520 | 2012-03-19 | ||
| PCT/EP2013/053510WO2013139554A1 (en) | 2012-03-19 | 2013-02-22 | Gram staining method with improved decolorization of the crystal violet-iodine complex from gram negative bacteria |
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810139303.XADivisionCN108181155B (en) | 2012-03-19 | 2013-02-22 | Gram staining method with improved decolorization of crystal violet-iodine complex of gram negative bacteria |
| Publication Number | Publication Date |
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| CN104245955Atrue CN104245955A (en) | 2014-12-24 |
| CN104245955B CN104245955B (en) | 2018-03-16 |
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